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 Aeglidae
Life History and Conservation Status of
Unique Freshwater Anomuran Decapods
 Advances in Crustacean Research
Ingo S. Wehrtmann
University of Costa Rica, San Jose
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The Biology of Squat Lobsters
edited by Gary C.B. Poore, Shane T. Ahyong, and Joanne Taylor
Aeglidae: Life History and Conservation Status of Unique Freshwater Anomuran Decapods
edited by Sandro Santos and Sergio Luiz de Siqueira Bueno

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 Aeglidae
Life History and Conservation Status of
Unique Freshwater Anomuran Decapods

Edited by
Sandro Santos and Sergio Luiz de Siqueria Bueno
CRC Press
Taylor & Francis Group
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Library of Congress Cataloging-in-Publication Data

Names: Santos, Sandro, editor.

Title: Aeglidae : life history and conservation status of unique freshwater
anomuran decapods / edited by Sandro Santos and Sergio Luiz de Siqueira
Bueno.
Description: Boca Raton : Taylor & Francis, [2020] | Includes
bibliographical references and index. | Summary: “Aeglidae focuses on
these unique crustaceans who are endemic to South America. The book is
the first to summarize the diverse aspects of the Aeglidae, whose
taxonomic features and phylogenetic relationships, evolutionary history
and biogeographical background, biological characteristics, and current
conservation awareness make them stand out among all other decapods.
This will be an important reference not only for carcinologists working
with this family of decapods, but also readers interested in the
evolution, biogeography, taxonomy, phylogenetics, physiology, and
reproductive ecology”-- Provided by publisher.
Identifiers: LCCN 2019025182 (print) | LCCN 2019025183 (ebook) | ISBN
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Subjects: LCSH: Aeglidae.
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 Contents
Preface......................................................................................................................vii
Editors........................................................................................................................ix
List of Contributors....................................................................................................xi

Chapter 1
Evolutionary History and Phylogenetic Relationships of Aeglidae............................ 1
Marlise Ladvocat Bartholomei-Santos, Sandro Santos, Bianca Laís
Zimmermann, Marcos Pérez-Losada, and Keith A. Crandall

Chapter 2
Morphology, Taxonomy, and Diversity of Extant Aeglidae..................................... 29
Sandro Santos, Georgina Bond-Buckup, and Carlos G. Jara

Chapter 3
Population Structure and Morphological Maturity.................................................. 73
Setuko Masunari

Chapter 4
Trophic Ecology........................................................................................................97
Pablo Collins

Chapter 5
Reproductive Biology and Gonadal Development in Aeglidae.............................. 133
Carolina Sokolowicz

Chapter 6
Postembryonic Development, Parental Care, and Recruitment.............................. 155
Sérgio Luiz de Siqueira Bueno, Roberto Munehisa Shimizu,
and Juliana Cristina Bertacini Moraes

Chapter 7
Intra- and Interspecific Behavioral Interactions of Aeglidae with a
Comparison to Other Decapods.............................................................................. 181
Marcelo M. Dalosto and Alexandre V. Palaoro

v
vi Contents

Chapter 8
Physiological Ecology: Osmoregulation and Metabolism of the Aeglid
Anomurans..............................................................................................................203
John Campbell McNamara and Samuel Coelho Faria

Chapter 9
Conservation Status and Threats of Aeglidae: Beyond the Assessment................. 233
Harry Boos, Paula Guimarães Salge, and Marcelo A. A. Pinheiro

Chapter 10
Sampling and Data Analysis for Population Studies on the Life History of
Aegla spp................................................................................................................. 257
Roberto Munehisa Shimizu and Sergio Luiz de Siqueira Bueno
Index....................................................................................................................... 279
 Preface
A preface to a unique taxon: Aegla Leach, 1820, a crown jewel among South
American freshwater decapods.
This book is about one single taxon: Aegla Leach, 1820. And what a remarkable
taxon it is! Those who had—and those who are having—the experience of study-
ing these unique freshwater decapods could not agree more with Schmitt’s remarks
written down on the first page (Schmitt, 1942; p. 431) in his seminal monography on
aeglids: “There are no freshwater Crustacea at all like Aegla anywhere else in the
world.”
The production of this book comes in a special moment because we find our-
selves at the brink of celebrating 200 years since the first taxonomic entry of an
extant aeglid, as Galathea laevis, in the scientific literature. Over these two cen-
turies, hundreds of investigations on Aegla have been published. A brief search for
Aeglidae on Google Scholar, for example, retrieves more than 1,600 entries.
Aegla is the only taxon within the Anomura whose representatives are entirely
adapted to the freshwater environment. As of 2018, there are now 87 known valid
species, all endemic to subtropical and temperate South America. This figure makes
Aegla the most species-rich genus of all true freshwater decapods in this subconti-
nent. The tally is certainly bound to go up considerably as putative new species are
being recognized and still waiting for the necessary formal description (Chapter 1),
and as unexplored or poorly explored areas within the known range of distribution
continues to be systematically investigated. It is only reasonable to expect that the
number of valid species may soon surpass the barrier of 100 species within the next
few years ahead.
This book is also about perhaps the most endangered freshwater decapod in the
Neotropical Region (Chapter 9). About 70% of the 87 known species are currently
threatened with extinction, having been assessed as critically endangered, endan-
gered, or vulnerable threatened categories, as defined by the International Union for
Nature Conservation. The main threats to aeglids include the removal of riparian
forest, habitat fragmentation and destruction, industrial, agricultural, livestock, and
domestic pollution of the water bodies.
One unique feature about Aegla is the fact that its evolutionary history can be
told based on sound scientific evidence, starting from marine fossil representatives
to the successful adaptation of Aegla to freshwater habitats and the subsequent dis-
persal routes through paleobasins of continental South America that neatly explain
the distributional pattern we see today (Chapter 1). The successful adaptation to
freshwater environments demanded the acquisition of adaptive life history strate-
gies, most importantly those regarding physiological ecology (Chapter 8), postem-
bryonic development and parental care (Chapter 6).
Morphological studies have been a strong line of investigation starting right from
the beginning. Schmitt’s monography (1942) may still be the most revered landmark
publication on the taxonomy of Aegla, but other South American leading investiga-
tors have published several equally important papers on this topic since the 1980s

vii
viii Preface

(see Chapters 1 and 2 for references therein). Together, this bulk of publications on
aeglid taxonomy has provided a great contribution to the knowledge of Aegla distri-
bution and diversity. More recently, molecular analyses have made a huge impact in
systematic studies of aeglids, providing valuable insights and hypotheses regarding
the phylogenetic relationships among Aegla species as well as the phylogenetic posi-
tion of the family Aeglidae within the Anomura (Chapter 1).
Throughout the pages of this book, the reader will also have the opportunity
to check out fine compilations on topics such as population structure and matu-
rity (Chapter 3), trophic ecology (Chapter 4) as well as reproduction and gonadal
development (Chapter 5) and behavior (Chapter 7). Finally, Chapter 10 deals with
sampling techniques, handling procedures, and provides a discussion on analytical
treatments of data obtained under field working conditions.
For us, the editing experience involved in the production of this book has been a
quite extraordinary one. We are really grateful to our colleagues Dr. Ingo Wehrtmann
and Dr. Célio Magalhães for having invited us to carry out this task, which we hum-
bly accepted without hesitation. Also, we wish to demonstrate our gratitude to all
who have directly or indirectly contributed to this book. We thank all authors of the
chapters: Alexandre V. Palaoro, Bianca Laís Zimmermann, Carlos G. Jara, Carolina
Sokolowicz, Georgina Bond-Buckup, Harry Boos, John Campbell McNamara,
Juliana Cristina Bertacini Moraes, Keith A. Crandall, Marcelo A. A. Pinheiro,
Marcelo M. Dalosto, Marcos Pérez-Losada, Marlise Ladvocat Bartholomei-Santos,
Pablo Collins, Paula Guimarães Salge, Roberto Munehisa Shimizu, Samuel Coelho
Faria, and Setuko Masunari. We are also especially grateful to the researchers
who kindly collaborated with us reviewing the chapters: Antônio Leão Castilho,
Christopher Tudge, Ingo Wehrtmann, Marlise L. Bartholomei-Santos, Marcos
Tavares, Neil Cumberlidge, Roberto Shimizu, and Rodney Feldmann.

Sandro Santos and Sérgio Bueno

May 2019

REFERENCE

Schmitt, W. 1942. The species of Aegla, endemic South American freshwater crustaceans.
Proceedings of the United States National Museum 91:431–520.
 Editors

Sandro Santos is a full professor of the Department of Ecology and Evolution at

the Universidade Federal de Santa Maria, Brazil. He holds a Ph.D. in zoology from
Universidade Estadual Paulista (UNESP), a post-doctorate in systematics of Aeglidae
from Universidade Federal do Rio Grande do Sul (UFRGS) and phylogeography of
Aeglidae from George Washington University (GWU). For twenty years he has been
studying aeglid crustaceans, focusing on population structure, reproductive biology,
behavioral ecology, systematics, and conservation.

Sérgio Luiz de Siqueira Bueno is associate professor at the Department of Zoology,

Institute of Bioscience, University of São Paulo, Brazil. He has dedicated the past
15 years of his research career to the study of aeglids. These studies are focused
on the relative growth, reproduction, life cycle, postembryonic development, taxon-
omy, estimation of population size, and conservation of these remarkable freshwater
decapods.

ix
 List of Contributors
Marlise Ladvocat Bartholomei‑Santos John Campbell MacNamara
Departamento de Ecologia e Evolução Departamento de Biologia - Faculdade
Universidade Federal de Santa Maria de Filosofia, Ciências e Letras
Santa Maria, Brazil Universidade de São Paulo
Ribeirão Preto, Brazil
Georgina Bond-Buckup
Departamento de Zoologia Setuko Masunari
Universidade Federal do Rio Grande Departamento de Zoologia
do Sul Universidade Federal do Paraná
Porto Alegre, Brazil Curitiba, Brazil

Harry Boos Juliana Cristina Bertacini Moraes

Instituto Chico Mendes de Conservação Departamento de Zoologia
da Biodiversidade Universidade de São Paulo
Itajaí, Brazil São Paulo, Brazil
Pablo Collins
Alexandre Palaoro
Facultad de Humanidades y Ciencias
Departamento de Ecologia
Universidad Nacional del Litoral
Universidade de São Paulo
Santa Fé, Argentina
São Paulo, Brazil
Keith A. Crandall
Computational Biology Institute Marcos Pérez‑Losada
George Washington University Computational Biology Institute
Ashburn, Virginia George Washington University
Ashburn, Virginia
Marcelo Dalosto
Departamento de Ecologia e Evolução Marcelo A. A. Pinheiro
Universidade Federal de Santa Maria Instituto de Biociências
Santa Maria, Brazil Universidade Estadual Paulista
São Vicente, Brazil
Samuel Faria
Departamento de Fisiologia Geral Paula Guimarães Salge
Instituto de Biociências Instituto Chico Mendes de Conservação
Universidade de São Paulo da Biodiversidade
São Paulo, Brazil Itajaí, Brazil

Carlos G. Jara Sandro Santos

Instituto de Zoología Departamento de Ecologia e Evolução
Universidad Austral de Chile Universidade Federal de Santa Maria
Valdivia, Chile Santa Maria, Brazil

xi
xii List of Contributors

Roberto Munehisa Shimizu Carolina C. Sokolowicz

Departamento de Ecologia Departamento de Zoologia
Universidade de São Paulo Universidade Federal do Rio Grande
São Paulo, Brazil do Sul
Porto Alegre, Brazil
Sérgio Luiz de Siqueira Bueno
Departamento de Zoology Bianca Laís Zimmermann
Universidade de São Paulo Instituto Federal de Educação Ciência e
São Paulo, Brazil Tecnologia
Ibirubá, Brazil
 Chapter  1

Evolutionary History and Phylogenetic

Relationships of Aeglidae

Marlise Ladvocat Bartholomei-Santos, Sandro Santos, Bianca Laís

Zimmermann, Marcos Pérez-Losada, and Keith A. Crandall

CONTENTS

1.1 Evolutionary History: Marine Ancestors and Continental Invasion................. 1

1.2 Phylogenetic Relationships................................................................................7
1.3 Aegla Speciation and Dispersion in Freshwater Environments....................... 16
1.4 Cryptic Species in Aegla.................................................................................. 19
1.5 Perspectives.....................................................................................................20
References................................................................................................................. 21

1.1 EVOLUTIONARY HISTORY: MARINE ANCESTORS

AND CONTINENTAL INVASION

The family Aeglidae Dana, 1852 has had a fairly successful history of diversifi-
cation within South American fresh waters. The family comprises only one extant
genus, Aegla, with 87 known species (Bueno et al. 2017; Santos et al. 2017; Jara
et al. 2018; Páez et al. 2018) and many others to be described (Crivellaro et al. 2017;
Zimmermann et al. 2018). But how and when did this history start? Why has this
anomuran group succeeded in fresh water? Although we still do not have complete
answers for many aspects of Aeglidae evolution, and numerous points remain to be
elucidated, several studies have shed light on many of these issues.
The family Aeglidae belongs to the infraorder Anomura, which has succeeded in
colonizing a variety of ecosystems, including marine, brackish, terrestrial, freshwa-
ter, and hydrothermal vent habitats (Bracken-Grissom et al. 2013). Aegla has its life
cycle entirely restricted to freshwater environments. Beyond aeglids, only a single
species of Diogenidae, the hermit crab Clibanarius fonticola, is known to perma-
nently inhabit freshwater habitats (McLaughlin and Murray 1990).

1
2 Aeglidae

Currently, the family Aeglidae is distributed across rivers, streams, and lakes
in multiple basins flowing to both the Atlantic and Pacific coasts of southern South
America, between the latitudes of 20°18ʹS in Brazil (Bueno et al. 2007) and 50°34ʹS
in Chile (Oyanedel et al. 2011). Although the extant aeglids are all living in fresh
water, the family originated in the marine environment, as revealed by two fossil
species of Aeglidae (Feldmann 1984; Feldmann et al. 1998).
Before the description of the first fossil member of the family Aeglidae (see
Feldmann 1984), some naturalists had already speculated about its origin and dis-
persion within the continent. Schmitt (1942, p. 443) stated that “the marine origin
of Aegla appears indisputable.” Mentioning that less ornamented species could be
more primitive, Schmitt (1942) hypothesized that Aegla jujuyana would be the clos-
est taxon to the Aegla ancestor. This would place the center of distribution of the
genus in the Province of Jujuy, in northwestern Argentina. From there, variants with
the “Pacific type of rostrum” (flatter and troughed) would have spread out westward
to the Andes and Chile and eastward to the Serra do Mar in Brazil, while the forms
with the “Atlantic type of rostrum” (spine-like and ridge-roofed) dispersed through-
out the Paraná River and Uruguay River basins. Based on information provided by
an article about the geology of South America (Berry 1922), Schmitt (1942) also
speculated that since Jujuy had a marine history, with marine deposits antedating
the Devonian, up to the Carboniferous, the gradual elevation of the land above the
sea level allowed the Aegla ancestors to adapt progressively to less and less salty and
increasingly fresh water; from the Cretaceous, the Jujuy region would have been
totally continental, and its waters would be no longer marine. Aegla franca (found in
São Paulo state, Brazil) would not fit into this scenario due to its intermediate type
of rostrum, more similar to that of A. jujuyana. Thus, the first species could be a
northeastern offshoot of the ancestral or original jujuyana stock (Schmitt 1942). As
the opening of the South Atlantic Ocean is accepted to have occurred progressively
from south to north starting in the early Jurassic (reviewed in Seton et al. 2012), one
can infer that if Schmitt (1942) was correct in his speculations, the aeglid ancestor
would have come from the Pacific Ocean occupying the Jujuy region in Argentina
in the past; however, he did not mention either a Pacific or an Atlantic origin for
Aeglidae in his monograph. Ringuelet (1948) disagreed with Schmitt’s view and
pointed out that accepting Jujuy as the dispersion center would require descending
to the Paleozoic to find marine sediments, and that would be much too old to find
the marine ancestors of the extant aeglids. In fact, the oldest known anomuran fossil
dates from the Triassic (Chablais et al. 2011). It is worthy to note that the Atlantic and
Pacific types of rostra identified by Schmitt (1942) occur in both South American
coasts among the currently described species. Hence, Schmitt’s classification of aeg-
lids based on rostra does not hold. Actually, Ringuelet (1948, 1949a) subsequently
disagreed with Schmitt observing that there are different intermediate types of ros-
tra. Moreover, he hypothesized that the most primitive species of Aegla would have
a prominent rostrum, with wide extra-orbital sinus; from this type, there would be
two possible evolutionary scenarios, one leading to species with an elevated (cari-
nated), but short rostrum, with somewhat obtuse, somewhat excavated carina and a
narrow extra-orbital sinus; or another leading to species with short, depressed rostra
Evolutionary History and Phylogenetic Relationships of Aeglidae 3

(non carinated) with a narrow extra-orbital sinus. Both scenarios would lead to the
complete disappearance of the orbital spines and extra-orbital sinuses. Based on this
idea, Ringuelet (1948, 1949a) also rejected the primitive status of Aegla jujuyana
proposed by Schmitt (1942).
Forty years before Schmitt (1942), Ortmann (1902) had already hypothesized
a possible Pacific origin for aeglids by comparing the distributions of Aegla and
Parastacus Huxley, 1879, in South America. He speculated that as the family
Parastacidae was present in Australia in the Upper Cretaceous, it could have spread
into Antarctica and southern Chile, and in the early Tertiary into Northern Argentina
and Southern Brazil. Since the genus Aegla had a close distribution to Parastacus,
the pathway could have been similar for aeglids, although Ortmann (1902) believed
that an Antarctic origin was improbable for the latter group, and did not discard the
possibility of the inverse path, that is, from the Atlantic to the Pacific side. Moreover,
he pointed out that the presence of the genus Aegla (as well as Parastacus) on both
sides of the Andean Cordillera indicates that this distribution predates the complete
uplift of the Andes, as the mountain chain would act as a barrier for their dispersion.
Schmitt (1942) partially disagreed with this idea, considering that Aegla might not
have had a wide distribution before the Andes reached their present height because
passages in the Chilean and Argentinean lake regions could have allowed dispersion.
Later, by using a panbiogeographic analysis, Morrone and Lopretto (1994) suggested
a single generalized track oriented from northeast to southwest, indicating the pre-
existence of ancestral biotas, based on the congruence of individual tracks for three
freshwater decapod groups (Aeglidae, Parastacidae, and Trichodactylidae). Their
analysis identified Aegla uruguayana from the Atlantic side as the most primitive
species.
The discovery of a fossil member of the family Aeglidae, Haumuriaegla glaess-
neri, from marine sediments in New Zealand corresponding to the Haumurian stage
in the Late Cretaceous (Feldmann 1984), has definitively supported the marine ori-
gin for the group. Feldmann (1984) suggested that the family could have originated
in the Indo-Pacific region and dispersed eastward, reaching South America. The
dispersion possibly occurred before the end of the Oligocene, preceding the sepa-
ration of Australia and New Zealand from Antarctica and the development of the
circum-Antarctic current system (Feldmann 1986). The circumpolar current isolated
Antarctica, blocking the heat transfer from the low latitudes and allowing glaci-
ation to develop (reviewed in Martin 2006), contributing to the isolation of New
Zealand from South America in post-Oligocene time (Feldmann 1986). Although
the extant aeglids do not present a larval phase, a possible adaptation to a fresh-
water life cycle (Pérez-Losada et al. 2002a; McLaughlin et al. 2007), their marine
ancestors may have spread through larval dispersal in the main counter-clockwise
gyre of ocean circulation in the southern Pacific Ocean (Feldmann 1986). An even
older fossil from a member of the family Aeglidae, dating from the Albian stage in
the Early Cretaceous, was found in Mexico (Feldmann et al. 1998). The descrip-
tion of Protaegla miniscula (Feldmann et al. 1998) not only supported the hypoth-
esis of a Pacific origin for the group but also added a third genus to the family
Aeglidae. Therefore, we currently accept two marine fossil genera (Haumuriaegla
4 Aeglidae

and Protaegla) and one extant freshwater genus (Aegla) for the aeglids. The oldest
known anomuran fossil is from the Late Triassic (Chablais et al. 2011), and most ano-
muran superfamilies were already present in the Jurassic fossil record, so the origin
of the family Aeglidae could be earlier than its oldest known fossil (Pérez-Losada
et al. 2004).
At the transition between the Cretaceous and the Paleogene, many taxa expe-
rienced a mass extinction; a phenomenon commonly referred to as Cretaceous-
Paleogene (K/Pg) event (Renne et al. 2013). The K/Pg event does not seem to
have affected all taxa similarly, since some decapod families show high survival
rates across the K/Pg boundary (Schweitzer and Feldmann 2005). The geographic
distribution was an essential factor to cross the boundary, and decapod genera
inhabiting temperate and high-latitude areas had higher survival rates than lower
latitude genera (Schweitzer and Feldmann 2005). Although the two aeglid fossil
genera apparently did not cross the boundary, the family survived into the pres-
ent. Schweitzer and Feldmann (2005) hypothesized that the family Aeglidae could
have survived because it was either a refugium taxon or inhabited a buffered habi-
tat. Refugia taxa could migrate to secondary habitats not as impacted by the event
causing the mass extinction; most of them represent species or their descendants
that have been forwarded to more restrained habitats by competitive displace-
ment from their marine environments (Harries et al. 1993). On the other hand,
buffered habitats were not greatly disturbed by the mechanisms causing the mass
extinction, as could be the case in temperate and high-latitude regions (Harries
et al. 1993). The marine ancestor of the extant aeglids could have originated in the
high southern latitudes during the Cretaceous (Feldmann and Schweitzer 2006)
or earlier.
An alternative way to investigate the question of a Pacific or Atlantic origin of
Aeglidae is by means of a phylogeny of the extant species. The basal taxa would
be the first ones to diverge, and their area of occurrence (if in the Atlantic or the
Pacific side) would indicate the possible path by which a marine ancestral might
have invaded the continental waters. However, the conservative general morphotype
and the low number of shared apomorphic characters relative to the large number
of extant species (Bond-Buckup and Buckup 1994), coupled with the presence of
homoplasic characters, present challenges to building a morphology-based phylog-
eny for Aeglidae. In situations like this, molecular approaches are very useful to
elucidate the evolutionary history of a group of organisms by increasing the num-
ber of characters available for analysis. Initially, a molecular phylogeny was built
using four mitochondrial genes from 17 Chilean aeglid species, two trans-Andean
Aegla species collected in Argentina, with one galatheid and one porcellanid spe-
cies used as outgroups (Pérez-Losada et al. 2002a). Aegla papudo from Chile stood
in a basal position, as the sister group of the other Aegla species in the phylogeny,
supporting a Pacific origin for Aeglidae. The basal position of A. papudo within
the aeglids was also confirmed by constructing phylogenies using four mitochon-
drial and two nuclear genes and five anomuran members as outgroups (Lomisidae,
Porcellanidae, Chirostylidae, Galatheidae, and Paguroidea), with three different
Evolutionary History and Phylogenetic Relationships of Aeglidae 5

approaches: Maximum Likelihood (ML), Maximum Parsimony, and Bayesian (BA)

analyses (Pérez-Losada et al. 2004).
Due to computational limitations, Pérez-Losada et al. (2004) used A. papudo as
a functional outgroup to investigate the phylogenetic relationships among 58 out of
63 known Aegla species at the time and six undescribed species. Their ML and BA
consensus trees placed A. ringueleti as the most basal ingroup taxon and A. scamosa
in the next most basal position. The remaining taxa, except A. marginata and A.
spinipalma, were clustered into five major clades, named as clades A, B, C, D, and
E. Clade A was the first to diverge, followed by clade B, both encompassing species
from Chile and southern Argentina. The tree splitting pattern suggested an eastward
radiation for Aeglidae. An alternative westward radiation hypothesis or two indepen-
dent radiations in both directions were also tested and rejected by the authors. Thus,
Pérez-Losada et al. (2004) supported the Pacific origin hypothesis, in agreement
with fossil evidence (Feldmann 1984; Feldmann et al. 1998). Moreover, the age of
the most basal node in the ML tree was estimated as 74 million years ago (Mya), also
in accordance with the fossil data, which indicated that the Aegla relatives were at
least 75 My old (Pérez-Losada et al. 2004).
By integrating the history of South American drainages and phylogenetics,
Pérez-Losada et al. (2004) proposed a phylogeographic hypothesis to explain the
Aeglidae radiation. The species arrangements in the phylogenetic trees for the genus
Aegla do not perfectly match the current bifurcating pattern of river systems where
these species occur, which was not surprising since the present drainages were estab-
lished approximately 8 Mya (Potter 1997; Lundberg et al. 1998). The uplift of the
Andean Cordillera and the opening of the Atlantic Ocean significantly shaped the
Paleodrainage history in southern South America (Potter 1997; Lundberg et al. 1998;
Ribeiro et al. 2006). Most drainages from the western continental shields flowed
westward into the Pacific Ocean during the Jurassic and Early Cretaceous (Coney
and Evenchick 1994; Potter 1997). The drainage pattern for the South America plat-
form and adjacent Africa for that time is not well known, but large main streams
may not have been present since the region was probably arid or semi-arid (Potter
1997). The rifting in the South Atlantic was associated with substantial intracon-
tinental deformation within South America (Seton et al. 2012), as broad uplifts
along the southeastern Brazilian coast, causing an inward and coast-parallel flow
of Gondwanic drainages into the current La Plata River Estuary (Potter 1997;
Lundberg et al. 1998). In the far southern paleodrainages of Patagonia (southwestern
Gondwana), where the rifting began in the Mesozoic (~190 Mya, Jurassic period;
Seton et al. 2012), the Magallanes and Neuquén basins were connected to the Paleo-
Pacific, but starting in the Late Cretaceous, the paleo-rivers Colorado and Negro
inverted the flow into the Atlantic (Potter 1997). Also in that period, the uplift of
the Andean proto-cordillera caused the inversion of the flow in western drainages
from westward to eastward (Coney and Evenchick 1994; Potter 1997). From the Late
Cretaceous to the Early Paleocene, two large marine transgressions from the Pacific
occurred, from the North through the underfilled foreland basin and parallel to the
thrust front, reaching a river flowing northward in northwestern Argentina, between
the Sierras Pampeanas Massif and the uplifting Andes (Potter 1997; Sempere et al.
6 Aeglidae

1997; Lundberg et al. 1998; Bloom and Lovejoy 2011). In addition, two less extended
marine incursions from the South Atlantic penetrated the lower paleo-Paraná basin,
overlapping the Sierras Pampeanas Massif (Gayet et al. 1993) (Figure 1.1).
Pérez-Losada et al. (2004) suggested, by overlaying their phylogenetic hypoth-
esis onto the paleodrainage scenario, that the marine ancestor of the extant Aeglidae
radiated from the Pacific Ocean to the South American continent with one of the two
marine transgressions, that is, at least 60 Mya, when the second marine transgression
occurred. It is worthy to note that the introduction of aeglids into South America
took place only once, and the subsequent reproductive and physiological adapta-
tions to fresh water occurred in descendants of the ancestral invader population. The
clustering of the Argentinean species A. ringueleti and A. scamosa to the Chilean
species could be explained since the northward river flowing along the foreland basin

Figure 1.1 Distribution of Aegla species in southern South America, with an approximated

delimitation of the phylogenetic clades A to E from Pérez-Losada et al. (2004).
Named species are not included in any of the previous clades. (Modified from
Pérez-Losada et al. [2004] to encompass the new records of occurrence and the
species recently described.)
Evolutionary History and Phylogenetic Relationships of Aeglidae 7

was separated from the paleo-Paraná drainage by the Sierras Pampeanas Massif,
from the Late Cretaceous to the middle Eocene (Pérez-Losada et al. 2004). Several
modifications occurring in the western paleodrainages could have favored multiple
vicariance and migration events, producing the mixed pattern of present-day spe-
cies locations between clades A and B (Figure 1.1). The eastward propagation of
the Andean thrust front helped the Sierras Pampeanas to lose their influence as a
barrier from the Middle Eocene, and the western drainage between ~20o and ~35o S
was captured by the enlarging paleo-Paraná River (Lundberg et al. 1998), making
possible an eastward radiation of the Chilean aeglids (Pérez-Losada et al. 2004).
The significant uplift of the Andean Cordillera from the Late Oligocene (Sempere
et al. 1997) might have isolated the Chilean species from the eastern aeglids (Pérez-
Losada et al. 2004).
Clades C, D, and E radiated eastward following, to some extent, the pattern
of the current drainages primarily established in the Eocene (Potter 1997), where
the species occur: clade C over the Paraná River Basin, clade D over the western
tributaries of River Paraná and the Uruguay River Basin, and clade E over the
Guaíba River Basin (Pérez-Losada et al. 2004). The incongruences between some
phylogenetic clusters and the present-day distribution in drainage systems might be
connected to the paleodrainage changes (Pérez-Losada et al. 2004) occurring over
the last Tertiary periods, mainly the Paranan Sea and the uplifting of Serra do Mar
(Lundberg et al. 1998).
The update of the phylogenetic tree of the family Aeglidae, including the species
described since Pérez-Losada et al. (2004), integrated with new studies on South
America geological and hydrological history, will help to better clarify how this
unique group radiated within southern South America.

1.2 PHYLOGENETIC RELATIONSHIPS

The phylogenetic relationships of the family Aeglidae with their marine anomu-
ran relatives as well as among its species have long interested researchers. Initially,
the family Aeglidae was classified within the superfamily Galatheoidea. Latreille
(1818) drew for the first time an aeglid, previously unknown, without describing it,
which he named Galathea laevis. Schmitt (1942) speculated that Latreille might
have been unaware of the freshwater habitat of the species since he placed it in an
exclusively marine genus. Leach (1820) noticed that the specimen drawn by Latreille
(1818) represented, in fact, a new genus, naming it Aegla. Latreille (1829) highlighted
the similarities between the genus “Aeglea”* and the galatheids. Dana (1852) sepa-
rated the subtribe “Aegleidea” from the Galatheidea within the “Anomoura inferiora,”
and Girard (1855) graphed for the first time the name “Aegleidae” corresponding to
the current taxonomic level of the family. Schmitt (1942) believed that the closest

* According to Schmitt (1942), the misspelling Aeglea was introduced by Desmarest in 1825 and
followed by several authors (including Latreille) until Rathbun (1910) called attention to the first
orthography.
8 Aeglidae

relatives of the aeglids were marine and were to be found within the galatheids.
From that time on, aeglids have been included in the superfamily Galatheoidea by
most naturalists. However, the uniqueness of some characteristics of the aeglids led
to question this traditional view (Martin and Abele 1986; 1988). The Aeglidae is
the only anomuran family entirely restricted to fresh waters, it is endemic to south-
ern South America, and its gills and carapace sutures are different from those of
galatheids (Martin and Abele 1986, 1988). These and other features make the aeg-
lids unique ecologically, biogeographically, and morphologically (Martin and Abele
1986, 1988; Bond-Buckup and Buckup 1994).
Martin and Abele (1988) hypothesized a relationship between aeglids and her-
mit crabs (Paguroidea) due to similarities in some morphological characteristics,
but when the hypothesis was tested Aeglidae grouped with Galatheoidea instead of
Paguroidea in their analyses (Martin and Abele 1986). They proposed a phylogeny in
which aeglids would be the most primitive among Galatheoidea, with a sister-group
relationship between them (Martin and Abele 1986). The unique spermatozoal struc-
ture of Aegla provided some support to the elevation of the Aeglidae to the super-
family rank, besides suggesting a close affinity to Lomoidea (Tudge and Scheltinga
2002). A relationship between Aegla and Lomis was also suggested based on mito-
chondrial gene rearrangements (Morrison et al. 2002), along with morphological
and molecular data (Ahyong and O’Meally 2004; Porter et al. 2005). A Bayesian
tree constructed using an amino acid dataset from 13 protein coding mitochondrial
genes from 22 anomurans showed an unresolved relationship between Aegla and
Lomis, placing both in a basal position in relation to Chirostyloidea (Chirostylidae
and Kiwaidae), while the mitochondrial gene order analysis supported Aegloidea as
sister group to the clade formed by Lomisoidea and Chirostyloidea (Tan et al. 2018).
Based on a phylogenetic analysis of the nuclear 18S gene, Pérez-Losada et al.
(2002b) suggested the elevation of the family Aeglidae to the superfamily rank
due to its explicit separation from the galatheoid families. In their phylogenies,
Galatheoidea, excluding Aeglidae, presented a sister-relationship to Paguroidea with
Aeglidae being sister to the cluster Galatheoidea + Paguroidea. McLaughlin et al.
(2007) eventually proposed the superfamily Aegloidea based on a morphological
phylogeny. While the phylogenetic position of Aegloidea within the monophyletic
Anomura (Pérez-Losada et al. 2002b; Porter et al. 2005) has been controversial,
a recent study by Bracken-Grissom et al. (2013) clearly placed the Aeglidae as the
basal taxon in a strongly supported clade with Lomisidae, Eumunididae, Kiwaidae,
and then Chirostylidae branching off respectively.
The phylogenetic relationships within the Aeglidae have been speculated upon
since Schmitt (1942) suggested that A. jujuyana would be the most primitive spe-
cies, spreading out from the center of dispersion in Jujuy, Argentina, and giving
origin to species with the “Atlantic” and “Pacific” types of rostrum—except for A.
franca with an intermediate type of rostrum. Ringuelet (1948, 1949a) recognized
the difficulties in separating some species based on morphological characters due
to both the uniformity of the genus and intraspecific variation and even observing
that some specimens seemed to be hybrids. He reevaluated the taxonomic status of
Aegla affinis, allocating it as a subspecies of A. neuquensis (see Ringuelet 1948).
Evolutionary History and Phylogenetic Relationships of Aeglidae 9

It is interesting to observe that these two species represent a sister relationship in the
phylogeny of Pérez-Losada et al. (2004).
Ringuelet (1949a,b) considered A. parana, A. platensis, A. singularis, and A. uru-
guayana the most primitive species or the closest to the ancestral form. He split
the aeglids into five groups and presented a phylogenetic scheme for these groups
(Ringuelet 1949b). For matter of comparison, we present Ringuelet’s assemblages
in their corresponding clades (between parentheses) in the phylogenetic study of
Pérez-Losada et al. (2004) and in the present study in the case of A. franca and A.
paulensis (formerly A. odebrechtii paulensis). Group I encompassed A. singularis
(D) as the primitive species, originating* A. prado (D) and A. denticulata (A). Group
II had an unknown primitive species, but A. castro (C) as a stem species, origi-
nating A. franca (C) and, by a collateral branch, A. odebrechtii paulensis (C) and
finally A. odebrechtii (C). Group III also had an unknown primitive species, with A.
scamosa (no clade) as the stem species, originating A. neuquensis (A) and at last A.
neuquensis affinis (A); through another branch, A. scamosa would have originated
“A. spec.” from El Sosneado, in Mendoza, Argentina [later described as Aegla mon-
tana Ringuelet, 1960 and further considered junior synonymy with A. affinis (Bond-
Buckup and Buckup 1994)]. Group IV presented as the primitive species possibly A.
uruguayana (D), A. abtao (B) as the stem species, from which gave rise to A. abtao
abtao (B) and A. abtao riolimayana (B), subspecies that Ringuelet (1949b) proposed
based on its similarities. On another branch, A. abtao (B) would have originated
A. laevis (B), and this one would originate A. papudo (most basal species) and A.
concepcionensis (not evaluated). Group V encompassed A. parana (C) as the primi-
tive species, originating in a straight-line A. sanlorenzo (D), A. jujuyana (D), and A.
humahuaca (D). Some but not all of the affinities found by Ringuelet (1949b) are in
accordance with the current phylogeny of the aeglids.
Lopretto (1978, 1979, 1980, 1981) recognized four groups based on the fifth pair
of male pereopods: platensis group—A. platensis, A. singularis, A. uruguayana,
and A. neuquensis affinis (A. affinis); patagónico group—A. neuquensis neuquensis
(A. neuquensis) and A. abtao riolimayana (A. riolimayana); northwestern group—
A. humahuaca, A. franca, A. jujuyana, and A. sanlorenzo; cuyano group—A. mon-
tana (A. affinis) and A. scamosa. The groups “northwestern” and “cuyano” present
some similarities to the groups V and III of Ringuelet (1949b), respectively, but
Lopretto (1979) noticed large differences between A. neuquensis neuquensis and A.
neuquensis affinis concerning the studied appendix and questioned the validity of
the subspecies status.
Schuldt et al. (1988) proposed a preliminary cladogram based on morphological
characters for the species A. abtao abtao, A. abtao riolimayana, A. montana, A.
neuquensis affinis, A. neuquensis neuquensis, A. scamosa, and A. uruguayana from
central-western Argentina, in which A. uruguayana would be the most basal taxon.
Pérez-Losada et al. (2002a) presented a molecular phylogeny for 16 Chilean species,

* Ringuelet (1949b) used the verb “originar” (Spanish) in his study and we opted to keep his idea using
the English word “originate.”
10 Aeglidae

using four mitochondrial genes, placing A. papudo as the most basal aeglid species
and separating the remaining species in two major clades.
Pérez-Losada et al. (2004) performed the most complete study to date on the phy-
logenetic relationships within the Aeglidae. By using mitochondrial and nuclear genes,
they obtained robust trees for 58 species and six undescribed new species, in which
Aegla papudo from Chile was used as a functional outgroup after confirming its basal
position within the family. Aegla ringueleti was the most basal ingroup taxon, fol-
lowed by A. scamosa, both from western Argentina. The other species, except A. mar-
ginata and A. spinipalma, were grouped into five major clades. The species clustered
in each clade and the countries where they occur are shown in Table 1.1. Clade A (the
first to radiate) and clade B (the second to diverge) included the Chilean and southern
Argentinean species. Relationships among clades C, D, and E were not strongly sup-
ported, and alternative arrangements between these clades could not be rejected; they
encompassed the northern Argentinean, Uruguayan, and Brazilian species.
The study of Pérez-Losada et al. (2004) did not include five species known at the
time: Aegla concepcionensis and A. expansa, from Chile, and A. franca, A. lata, and A.
microphthalma from Brazil. The latter one is a stygobiotic species inhabiting a single
cave with difficult access (Bond-Buckup and Buckup 1994) and the other four species
could not be found at the time of the study. On the other hand, the authors included
six putative new species (named n. sp. 1 to n. sp. 6 in the tree), which were described
later under the names A. muelleri Bond-Buckup and Buckup, 2010 (Aegla sp. n. 1
and 5); A. pomerana Bond-Buckup and Buckup, 2010 (Aegla n. sp. 2); A. brevipalma
Bond-Buckup and Santos, 2012 (Aegla n. sp. 3); A. renana Bond-Buckup and Santos,
2010 (Aegla n. sp. 4); and A. saltensis Bond-Buckup and Jara, 2010 (Aegla n. sp. 6).
Moreover, 18 new species not included in the study of Pérez-Losada et al. (2004)
were also described after 2004: Aegla manuinflata Bond-Buckup and Santos, 2009;
Aegla leachi Bond-Buckup and Santos, 2012; Aegla oblata Bond-Buckup and Santos
2012; Aegla georginae Santos and Jara, 2012; Aegla ludwigi Santos and Jara, 2013;
Aegla leachi Bond-Buckup and Santos, 2013; Aegla carinata Bond-Buckup and
Gonçalves, 2014; Aegla lancinhas Bond-Buckup and Buckup, 2015; Aegla loyo-
lai Bond-Buckup and Santos, 2015; Aegla meloi Bond-Buckup and Santos, 2015;
Aegla japi Moraes, Tavares, and Bueno, 2016; Aegla jaragua Moraes, Tavares,
and Bueno, 2016; Aegla jundiai Moraes, Tavares, and Bueno, 2016; Aegla vanini
Moraes, Tavares, and Bueno, 2016; Aegla charon Bueno and Moraes, 2017; Aegla
quilombola Moraes et al. 2017; Aegla okora Páez and Teixeira, 2018; and Aegla
chilota Jara, Pérez-Losada, and Crandall, 2018. Also, the species Aegla rosanae,
Campos Jr., 1998, which was synonymized to Aegla paulensis (Bond-Buckup and
Buckup 2000), was revalidated by Moraes et al. (2016). Thus, the number of Aegla
species described to date is 87, although at least another 15 new species will be
described in the near future. Crivellaro et al. (2017) demonstrated that A. longirostri
encompasses a complex of 14 cryptic species; A. longirostri sensu stricto (from the
type-locality) and nine undescribed species grouped with species from clade E; the
remaining three species clustered with species belonging to clade D (Figures 1.2 and
1.3; Table 1.1). The widely distributed A. platensis encompasses three distinct spe-
cies (Zimmermann et al. 2018), all clustering with species from clade D (Figures 1.2
Table 1.1 Species Clustering in Major Clades (A to E) in the ML/BA Consensus Phylogeny of the Genus Aegla, According to Pérez-Losada
et al. (2004), and Placement of the Species Not Studied Before or Described After 2004 (in Bold) within These Clades
No Clade Clade A Clade B Clade C Clade D Clade E
Aegla papudo Aegla bahamondei Aegla riolimayana Aegla camargoi Aegla singularis Aegla muelleri
Aegla ringueleti Aegla occidentalis Aegla abtao Aegla paulensis Aegla platensis sensu Aegla leptochela
stricto
Aegla scamosa Aegla neuquensis Aegla spectabilis Aegla perobae Aegla rossiana Aegla inconspicua
Aegla marginata Aegla affinis Aegla araucaniensis Aegla parva Aegla uruguayana Aegla serrana
Aegla Aegla alacalufi Aegla pewenchae Aegla parana Aegla intercalata Aegla franciscna
spinipalma
Aegla manni Aegla laevis Aegla castro Aegla prado Aegla ligulata
Aegla hueicollensis Aegla talcahuano Aegla schmitti Aegla violacea Aegla obstipa
Aegla denticulata denticulata Aegla cholchol Aegla cavernicola Aegla humahuaca Aegla renana
Aegla denticulata lacustris Aegla rostrata Aegla strinatii Aegla saltensis Aegla itacolomiensis
Aegla leachi(?) Aegla pomerana Aegla septentrionalis Aegla plana
Aegla oblata(?) Aegla leptodactyla Aegla jujuyana Aegla grisella
Aegla jarai Aegla sanlorenzo Aegla inermis
Aegla brevipalma Aegla manuinflata Aegla longirostri
sensustricto
Aegla spinosa Aegla carinata Aegla georginae
Aegla odebrechtii Aegla platensis Aegla ludwigi
species complex 1
Aegla lancinhas Aegla platensis Aegla longirostri
species complex 2 species complex 1
Aegla loyolai Aegla longirostri Aegla longirostri
species complex 10 species complex 2
Aegla meloi Aegla longirostri Aegla longirostri
Evolutionary History and Phylogenetic Relationships of Aeglidae

species complex 11 species complex 3

(Continued)
11
Table 1.1 (Continued) Species Clustering in Major Clades (A to E) in the ML/BA Consensus Phylogeny of the Genus Aegla, According to
12

Pérez-Losada et al. (2004), and Placement of the Species Not Studied Before or Described After 2004 (in Bold) within
These Clades
No Clade Clade A Clade B Clade C Clade D Clade E
Aegla franca Aegla longirostri Aegla longirostri
species complex 12 species complex 4
Aegla japi Aegla longirostri Aegla longirostri
species complex 13 species complex 5
Aegla jaragua Aegla longirostri
species complex 6
Aegla jundiai Aegla longirostri
species complex 7
Aegla vanini Aegla longirostri
species complex 8
Aegla rosanae Aegla longirostri
species complex 9
Aeglidae
Evolutionary History and Phylogenetic Relationships of Aeglidae 13

Figure 1.2 Bayesian tree based on 16S-COI haplotypes from a subset of species represent-
ing each phylogenetic clade of Pérez-Losada et al. (2004) and including species
recently described (in bold). Clades A to E are highlighted according to Pérez-
Losada et al. (2004). Bayesian posterior probabilities > 0.75 are shown above the
branches.
14 Aeglidae

Figure 1.3 Bayesian tree based on 16S sequences from a subset of species representing
each phylogenetic clade of Pérez-Losada et al. (2004) and including species
recently described (in bold). Clades A to E are highlighted according to Pérez-
Losada et al. (2004). The asterisk denotes species “misplaced” in relation to
the 16S-COI tree. Bayesian posterior probabilities > 0.75 are shown above the
branches.
Evolutionary History and Phylogenetic Relationships of Aeglidae 15

and 1.3; Table 1.1). We also present evidence for six new species (see following para-
graph). After the description of these species, the total number of species of the
genus Aegla will exceed 100.
The species A. japi, A. jaragua, A. jundiai, A. lancinhas, A. paulensis, A. rosa-
nae, and A. vanini belong to the A. paulensis complex, all of them occurring in
southeastern Brazil (Moraes et al. 2016). The specimens of A. paulensis used by
Pérez-Losada et al. (2004) were from the recently described A. jundiai, belonging to
clade C. In addition, Moraes et al. (2017) revised the taxonomic status of A. margin-
ata, splitting it into two species, one redescribed from the type locality and another
described as a new species, A. quilombola, from the Ribeira de Iguape Basin (São
Paulo state, Brazil). Aegla marginata was paraphyletic in the phylogenetic analysis
of Pérez-Losada et al. (2004), with specimens from the type locality not included in
any major clade, and specimens from the Ribeira de Iguape Basin clustering in clade
E (current A. quilombola), with a very close sister-relationship to A. leptochela, and
these two species being sympatric in one cave.
We obtained mitochondrial gene sequences (16S rRNA and COI) for the recently
described A. carinata, A. georginae, A. lancinhas, A. leachi, A. loyolai, A. lud-
wigi, A. manuinflata, A. meloi, A. oblata, and also for A. franca, not previously
included in the aeglid phylogeny (GenBank accessions FJ360714-15, FJ360706-
07, KT319222, KT319210, KT319218, KT319206, MH998634-63). Primer pairs
already described in the literature were used to amplify both genes (Pérez-Losada
et al. 2002a; Xu et al. 2009). Standard Polymerase Chain Reaction (PCR) was
conducted and PCR products were sequenced in both directions. Sequences were
aligned with Muscle (Edgar 2004). We performed phylogenetic analyses for sin-
gle and concatenated genes, using sequences both from all the species analyzed
by Pérez-Losada et al. (2004) and for a subset of species representing each clade
(AY050031-2; AY050035-6; AY0500042-4; AY050054-7; AY050065-6; AY050071-4;
AY050077-8; AY050081-2; AY050088-90; AY050100-3; AY050111-2; AY50117-20;
AY595549-51; AY595561; AY595565-7; AY595576-7; AY595581-2; AY595584-8;
AY595591-2; AY595594-9; AY595603; AY595605; AY595611-2; AY595623-4;
AY595627-32; AY595637-8; AY595641-6; AY595650-5; AY595658-9; AY595662-3;
AY595667-70; AY595803-5; AY595815; AY595819-21; AY595830-1; AY595835-6;
AY595839-42; AY595845-6; AY595848-53; AY595857; AY595859; AY595865-6;
AY595877-8; AY595881-6; AY595891-2; AY595895-900; AY595904-9; AY595912-3;
AY595916-7; AY595921-4; JQ844885-6; JQ844889; JQ844891-2; MF442421-2;
MF442424-5), and including sequences from undescribed new species of the cryp-
tic species complex A. longirostri (see Crivellaro et al. 2017) and A. platensis (see
Zimmermann et al. 2018), along with sequences from six putative new species. We
also conducted phylogenetic analyses including 16S rRNA sequences from A. japi, A.
jaragua, A. paulensis, A. rosanae, A. vanini, the only gene available in GenBank for
these species (GenBank accessions KU948368-73). The best-fit model of sequence
evolution selected by jModelTest 2.1.10 (Darriba et al. 2012) was GTR+Gamma+I for
all gene regions. Bayesian Inference was carried out using the Monte Carlo Markov
Chain method implemented in Beast 1.8.0 (Drummond et al. 2012). Analyses were
run for 30 million chains and sampled every 1000 generations. Posterior probabilities
16 Aeglidae

were calculated after a burn-in of three million states and checked for convergence
using Tracer 1.6 (Rambaut et al. 2014). Results were visualized using FigTree 1.4.2
(Rambaut 2014).
For better data visualization, we are showing the results for the analyses using
some representatives of each clade (Figures 1.2 and 1.3), instead of trees with all the
species, since the results did not differ between analyses. The clades within each
new species are inserted according to our analyses (Figures 1.2 and 1.3) and are also
shown in Table 1.1. Our concatenated 16S-COI tree recovered the same clades as in
Pérez-Losada et al. (2004), except that clade D was split into two different clades:
one clustering only northwestern Argentinean and a southern Bolivian species
from the Paraná River Basin, and the other including species occurring in southern
Brazil (Rio Grande do Sul state), Uruguay, southeastern Paraguay, and northeastern
Argentina. It is interesting to note that in the phylogenetic tree of Pérez-Losada et al.
(2004), clade D was split into two subclades and the species within each of them were
the same as in our tree (Figure 1.1). Another interesting finding was that two recently
described species from Rio Grande do Sul state, Brazil, A. leachi and A. oblata,
were clustered with species from clade B, from Chile, in a well-supported clade. A
phylogenetic tree constructed using 16S and COII mitochondrial genes (Santos et al.
2012) placed A. leachi within clade E and A. oblata within clade C.
The 16S tree produced mostly weakly supported clades (Figure 1.2) and did not
recover exactly the same clades as those in Pérez-Losada et al. (2004). Species from
clades A and B were mixed, and clades C, D, and E were split into two groups.
Species from the A. paulensis complex as well as A. franca clustered with some
species from clade C. Aegla leachi and A. oblata were grouped with species from
clade D. Although these clustering patterns may be easier to explain than those seen
in the concatenated tree, the support was low. By sequencing more genes for these
species, we will have a better understanding of their phylogenetic relationships. In
the 16S tree, A. georginae clustered with species from clade C while in our 16S-COI
tree as well as in a COII mitochondrial gene tree (Santos et al. 2013), it was placed
within clade E. Aegla parana, which was grouped within clade C in the multi-locus
tree of Pérez-Losada et al. (2004) and also in our 16S-COI concatenated tree and in
a concatenated 16S-COII tree (Santos et al. 2012), clustered with the northwestern
Argentinean species from clade D in the 16S tree. These results suggest that the
mitochondrial gene 16S alone may not be suitable to investigate phylogenetic rela-
tionships across all the Aeglidae.

1.3 AEGLA SPECIATION AND DISPERSION

IN FRESHWATER ENVIRONMENTS

The evolutionary history of Aegla is a formidable example of successful coloniza-

tion of freshwater habitats. The genus is represented by 87 described species, which
are found across Argentina, Bolivia, Brazil, Chile, Paraguay, and Uruguay. The distri-
bution of this impressive number of species includes all main rivers of southern South
America, except the southernmost Patagonian drainages (Bond-Buckup et al. 2008;
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 “I will, and more,” cried Sandie determinedly. “If he will not work, I am
but robbing you, and losing my own precious time besides.”
“Now, look here, Andrew: if there is any more of this, either now or any
other night, I’m going to give you a jolly good belting, and to-morrow I
shall bring a strap in my pocket for the purpose.” And so Sandie did, and
laid it ominously on the table in the boy’s sight.
Andrew became quite a reformed character after this, and Sandie used to
take him out for long rambles on Saturday afternoons, and to the church on
Sundays.
. . . . . .
A rather curious fact must here be mentioned regarding Willie Munro, as
it not only gives an additional insight to the lad’s character, but really has
some bearing on future acts in our story.
Willie, then, had never forgotten that fiasco on the hill, when he loaded
his gun by putting in the charge of shot first. He was a very sensitive boy,
and sometimes since that day, in his dreams, he would hear Maggie May’s
shrill peals of laughter, and see her merry mischievous face. Had Sandie
alone been there, it would not have mattered so much, but to have made a
fool of himself before a girl—ah! there was the rub. He felt at times that he
almost hated Maggie May, though surely it was no fault of hers.
However, he made a vow that he would rectify the mistake. He told his
father the whole story, and his father kindly acquiesced in his wishes. Willie
paid a visit to a keeper who lived a little way up Deeside. A crack shot he
was. The man was grooming a Lavereck setter when Willie reached his
humble dwelling.
“Fat can I dee for ye, laddie?” said Bob Brown, meaning, “What can I do
for you?”
“Oh,” said Willie, “I want you to teach me to shoot birds, so that I can go
to the hill and not make a fool of myself.”
Bob looked him all over. He even tested his eye-sight and the quickness
and steadiness of his hand.
“You’ll do,” he said. “How often can you come here?”
“Every afternoon, and I’m willing to pay fairly well.”
“Richt! in a month or sax weeks you ought to be as gweed (good) a shot
as mysel’. Hae ye a gun?”
 “No.”
“Weel, we’ll gang and buy ane the day.”
And so they did—not a very heavy one, but a breech-loader by one of
the best makers.
They also bought a spring-trap to throw crystal balls into the air, to
represent birds. These balls were filled with feathers, so it was easy to see
when they were broken.
For the first few days Willie was awkward enough, and hardly broke a
ball; then all at once he seemed, to get into the knack of the thing, and
broke the balls fast enough, and without apparent aim or effort.
The lad was rejoiced beyond measure. I am really afraid he neglected his
studies somewhat for this new-fangled fad of his, only he was determined to
wipe out what he looked upon as a stain on his character. He practised at
home every morning, as well as going to Bob’s in the afternoons.
Bob had a bit of private shooting, and now he began to take Willie out
with him, and an excellent hill-man the boy proved.
“Man,” said Bob more than once, “I’m perfectly prood o’ ye. And ye’re
a’ ma ain makin’ too.”
Willie now added the revolver to his armoury. Very awkward, indeed, he
was at first with this weapon, but the pistol was pronounced a good one,
and he soon became very precise in his shooting indeed.
Now Willie was sly.
Willie never told Sandie, his friend, what he was doing or studying. Not
he. If you had asked him why he did not, he might have replied—
“Because I know a trick worth two of that. I want my revenge. I want to
astonish Sandie, and Maggie May as well.”
There is a good old saying which, I must confess, has been of much
service to me during life. It is this: “You never know what you can do till
you try.”
I have often felt so ill, that I thought to get out of bed and begin literary
work would be a sheer impossibility. Then that bold saying has come to my
mind, and I have got up, and shaved myself—a terrible ordeal when one is
low and sick—and had my cold bath—another terrible ordeal, even for a
Scotsman, when out of form. Then I have had breakfast and begun work,
and wonderful to relate, the more I wrote the better I grew. What think you
of that, reader mine?
Well, in Willie’s case there was another proof of the truth of the grand
old aphorism. Willie persevered and persevered, and in six weeks’ time,
long before Christmas, he had been pronounced by Bob Brown a crack
shot, one who could single out his bird from a covey, and bring one down
with each barrel.
“I dinna think,” said Bob frankly and honestly, “I can teach you muckle
mair.”
But Willie went every night to Bob Brown’s all the same.
They had two spring-traps now, and two balls were dislodged into the air
at one time, and Bob rubbed his hands with delight, and laughed to see his
pupil smash each ball, making the feathers fly right and left.
. . . . . .
Sandie continued hard at his studies, especially mathematics, night after
night, and made considerable progress.
What a happy day that was, though, when his mother and sister Elsie
came to visit him.
Not only he, but Willie himself absented themselves from classes that
day, simply dropping a line to the various professors candidly owning up to
the cause of their playing truant.
So Sandie escorted his dear mother, and Willie chaperoned Elsie, all
over the Granite City. It was the first time Elsie had been to Aberdeen, and
she was naturally much struck by the marble whiteness of the stately
buildings.
The ladies were even taken into the quad to gaze upon the University at
which Sandie had achieved such signal success.
Then, when tired of wandering through the streets and seeing the lions of
the place, Willie—wilful Willie, as Sandie called him—insisted upon their
all dining together in the M‘Gregor Hotel.
“It is only four o’clock,” he said, “and you go away at six. Well, I would
have asked you to my house, but we will be ever so much more free and
easy here.”
“I shall pay,” said Sandie.
“Indeed, indeed you won’t.”
 “Oh, but I must.”
“Well, if you do, I shan’t come out with you to Kilbuie to spend the
Christmas week. So there!”
That settled it.
Not only did Willie pay, but he ordered the dinner, and it was one just
suited to the requirements of a bright clear winter’s day. No French names
either. 1. Delicious Scotch barley-broth. 2. Fresh salmon from the Dee,
caught the day before, not Norwegian salmon that had lain dead in ice for
three weeks, till all taste and flavour had fled to the moon or elsewhere. 3.
A juicy joint of roast-beef with snow-white mashed potatoes and
cauliflower. 4. Pudding and custard. 5. Cheese, oat-cakes, fresh butter, and
salad. For wine, although the ladies had their option, they chose good table-
ale, and the boys joined them. When about half-past five tea was brought, I
think both the mother and Elsie were very happy; at any rate, they both
confessed that they had never in their lives spent a more pleasant or happy
day.
“The time is getting short now,” said Mrs. M‘Crae, “and I want to make
sure of one thing.”
“And that is?” asked Willie.
“Sure o’ your promise to come out at Christmas when Sandie comes.”
“I promise, mother,” said Willie.
“You both look rather pale. I’m sure you’ve both been studying very
hard.”
Willie smiled inwardly, but made no reply.
They sauntered down to the station in good time, and just as they were
going away, and Elsie extended her hand to Willie, he gallantly pressed it to
his lips.
As he raised his cap, shy eyes met his, and a smiling but blushing face.
The whistle shrieked.
The train was gone.

CHAPTER IV

A STRANGE DUEL—BAD BOYS’ PRANKS

Some of the greatest treats Sandie enjoyed were his invitations out to
breakfast with his professors, some even whose classes he was not yet
attending inviting him. He could hardly have told you which of these he
liked best to breakfast with. There was old Dr. Brown, for example, who
filled the Greek chair, a very ugly but highly intellectual man, who spoke
like a Northumbrian, with a burr or rattle in the throat, and whom, as he
preferred the Doric dialect, the students had nicknamed “The Dorian.” The
Dorian, on ordinary days, used to finish his breakfast on the street, and
might be met in short cuts any morning eating a bap.[6] But on days when
he had students to breakfast, he was all there indeed, and up betimes. He
himself seemed blessed with the appetite of a Highland hunter, and he made
the students eat consumedly. But it was also a feast of reason and flow of
soul, and the number of racy anecdotes he told without apparent effort
during the breakfast-hour was marvellous; so too was the number of
buttered baps he got down.
Then there was Dr. Maclure, Professor of Humanity, that is, he filled the
Latin chair. A little man, perky, proud, and fat. He was an Englishman, but
a great admirer of Burns, whom he was constantly quoting. The students
called him “Cockie Maclure,” but it is to be hoped he did not know this.
However, breakfast with him, although not such a heavy meal as that with
the Dorian, was always most enjoyable.
Sandie used to think he would give a good deal could he only speak
English with so charming an accent.
Then there was poor Maxwell, so well known in the scientific world—
brown haired, handsome, thoughtful, and wise; he always had some
scientific marvel to tell his students about during breakfast. He was always
smiling, but never laughed a deal. I suppose he had an idea that strong tea
was not good for young fellows, for he invariably filled the cup half up with
rich delicious cream before pouring in the beverage.
Poor Maxwell! he is dead and gone, and great loss his death has been to
the world.
. . . . . .
Would my young reader fight a duel if called out? I should not advise
him to, though I myself have once or twice been foolish enough to appear
on the field and duly take my stand to shoot and be shot at.
 But in Sandie’s days duelling was not entirely unknown among the
students. One King’s College student sent his bullet through the left arm of
his opponent. Honour was declared to be satisfied after this, as well it might
have been.
Well, among Sandie’s intimate friends was a tall, pale-faced, aristocratic-
looking English lad named Coleman; a student our hero also knew was Tom
Brierly, a far more robust and daring-looking youth—a scapegrace, I fear.
At the University in the far North quarrels generate very simply sometimes.
For example, there lived with her mother in Upper Kirkgate a girl of about
seventeen. Sweet seventeen it was in her case, for she was very beautiful,
with eyes of darkest hazel, eyelashes that swept her cheeks, and a
complexion like strawberries and cream. Her mother and she made and sold
tuppenny pies. They did a good trade all day, but towards evening and up
till eleven o’clock that trade became a roaring one.
Well, Tom Brierly fell in love, or pretended to, with bonnie Mary
Mayne, and used to appear upon the festive scene every evening and eat
pies, till one could not have helped wondering how he could contain so
many. He also got Mary to teach him how to make them, and after he
became an adept he used to stand by her side and turn them out by the
dozen. For Tom was not a bit shy. On Sunday evenings the pair used to go
to church together if it rained, or out for a long walk if the weather was fine.
In fact, they were looked upon by all as sweethearts, and it was even
rumoured that Tom, who, by the way, was a clergyman’s son, was going to
marry Mary soon, and take up a pie-shop on his own account, which of
course would be doing infinite honour to his reverend daddie.
However, to make a long story short, who should Tom find one evening
when he paid his usual visit, but tall young Coleman, leaning over the
counter with a sickly smile on his face as he breathed sweet nothings and
the flavour of caramels in bonnie Mary’s face.
Tom wasn’t a man of many words, so he simplified matters and brought
them to an abrupt conclusion by seizing Coleman by his garments above
and below, and flinging him straight into the street. Coleman gathered
himself up.
“I cannot fight you with fists,” he said in a voice as like thunder as a
hen’s might be, “but a friend of mine shall call on you within an hour.”
And sure enough a friend did.
 Tom was laughing and joking with Mary, and turning out pie after pie
with extraordinary agility.
He hardly looked up.
“I won’t disappoint you,” he said; “keep your mind easy. I choose
pistols. My friend Smith, of 36 Union Terrace, will provide them. Yes,
seven o’clock, or say 7.30. We’d hardly see before. Go now and look Smith
up.”
And Tom coolly proceeded to turn out another pie. But poor Mary had
turned pale.
“You’re not going to fight—with—guns—are you, and all about me?”
“Keep your mind easy, Mary dear,” said Tom. “I don’t suppose we shall
hurt each other. And listen, Mary, I’ve made up my mind not to fire at his
head or body. I might let his little life out, you know. I mean to aim at those
thin legs of his.”
“Oh dear! oh dear!” mourned Mary, wringing her hands. “And where,”
she asked innocently, “will you fecht?”
“Oh,” replied Tom, as he rolled out a piece of paste, “there is only one
place. Smith knows it well, because I had a pugilistic encounter there with a
butcher. Round at the seaside of the Broad Hill. There won’t be a soul there
at that time of the morning. Pass the gravy, Mary.”
. . . . . .
It was some time past eleven o’clock. At the police-station near the
Tolbooth, a serjeant and one or two burly night-watchmen sat before a
roaring fire talking and laughing, when there entered a very pretty dark-
eyed maiden, with a shawl about her head. She appeared to be in very great
grief and trouble. But after she had told her story, she seemed comforted,
because in very kind tones the sergeant had replied—
“You keep your mind easy, my dear. Just go home and go to bed. We’ll
make it all right. Shall one of my men see you safe home?”
“Oh, no,” was the reply, “I’ll soon run home.”
. . . . . .
Tom and his second, Smith, were up and dressed even before the stars,
that had been shining so brightly all night, had commenced to pale before
the coming of day. Smith, after warming coffee, busied himself in getting
the irons ready.
 He was a brave, smart little fellow, Smith, and the idea of aiming at
Coleman’s thin legs tickled him very much, and made him laugh as he
cleaned the pistols.
“His thin legs, eh?” he said. “Well, friend Tom, you’ll be a smart shot if
you hit ’em. Why, it will be like firing at a couple of raspberry canes.”
A little after seven both young men started for the links and Broad Hill.
They got right up over the top of the hill, and having gained the summit,
looked beneath them. Yes, Coleman and his second were already there,
although the time was not yet up.
What a heavenly morning it was too! The sun was not yet up, but red
and crimson and golden clouds flecked all the eastern sky, and were
reflected from the rolling waves till all the ocean seemed ablaze. Only on
the yellow sands were the long lines of snow-white foam, where the seas
broke lazily upon the beach.
“What a pity,” said Tom with a sigh, “to have to face so deadly an
encounter on a morning like this!”
“I daresay,” said Smith, “if you were to apolo——”
He never got any further, Tom stopped him with a look.
Five minutes after this, Tom and his opponent had shaken hands, and
stood facing each other at twelve paces waiting for the words, “One, two,
three, fire!” when suddenly from behind a sandhill at no great distance
started two burly policemen. They appeared to spring from the very earth.
“Halt!” That was the stentorian word of command they gave.
“Boys!” cried Smith, “there has been a magpie about. Policemen,” he
added, “did you cry ‘halt’?”
“We cried ‘Halt!’ ”
“Then I cry something else, ‘Bolt!’ ”
He suited his own actions to the word, and before either of those
policemen could say “Jack-knife,” the race for liberty had commenced.
All honour to the bobbies; they did give chase, but as well might a
tortoise try to catch a weasel. They were speedily distanced and left
breathless far behind.
The four students went on to Balgownie Bridge, then crossed country to
Woodside, when, coming to a farm, they succeeded in breakfasting on curds
and cream, oatcakes, fresh butter, and new-laid eggs.
Both seconds declared that, under the circumstances, honour should be
deemed satisfied. Then both principals shook hand, each, declaring himself
in the wrong. Thus was a friendship established between Tom Brierly and
Coleman, and—and—and they lived happy ever afterwards. But this is the
true story of an Aberdeen University duel.
They never heard another word from the police-office about the
escapade, so rightly judged that the magistrates had forgiven them.
. . . . . .
My description of University life in the Granite City during Sandie’s
curriculum would be incomplete were I to say nothing of what I may call
the bad boys of the College. Of course, you find these everywhere, though
in after life they are sure to look back with some degree of sorrow on the
days that are gone never to return.
Tom Brierly was one of these. Sandie tried hard to reform him, but I fear
with little success.
Sandie more than once, thinking that example was better than precept,
accompanied Tom to Mother Robertson’s, an inn in the Guestrow, much
frequented by the students. There were more merry faces round the tables of
the coffee-room than there had any right to be; there were more steaming
tumblers of toddy, and there would be more headaches in the morning.
Sandie drank nothing but stone bottles of ginger-beer.
“How can you be so merry on that?” cried Tom.
“It’s all custom,” said Sandie. “I feel very happy and merry on this, and I
won’t have the ghost of a heavy head in the morning.”
So Sandie sat with them, and he told stories that made every one laugh,
and he sang songs that made some of them cry; but at ten o’clock he arose,
and, in spite of their importunities, bade all good-night and walked straight
home to his attic.
The principal practical jokes performed at night by the students in
Sandie’s day were extinguishing gas lamps, wrenching off knockers with
the twist of a strong stick, and pulling out bell-handles.
The night-watchmen, as they were called, were certainly a body of grand
men. Their physique left nothing to be desired. But then they were not
active.
 They were called “Charlies,” just as the day-policemen were
denominated “Bobbies.”
These sturdy fellows were dressed in strong broadcloth fear-nothing
coats, that reached down to their heels; they wore broad Tam o’ Shanter
bonnets, and were armed with oaken cudgels big enough to have felled an
ox.
At nine o’clock each evening they were marshalled two deep in front of
the watchhouse door. The officer gave the words of command in the
broadest of Scotch.
“Are ye a’ richt there, Jamie?” This to the sergeant.
“A’ richt, sir,” Jamie would reply.
“Weel, richt fut foremaist. Quick mairch! awa’ ye gang.”
And away they went, filing off here and there at the corners of streets to
take up their several beats.
But the bad boy students were the bane of those poor fellows’ lives.
There was no saying when one or two would turn up.
They would see lamp after lamp extinguished right ahead of them,
sometimes a whole street placed in darkness, and yet be powerless to give
chase to the light-footed lads.
Or they would hear sounds like shots fired in the quiet streets, bang!
bang! bang! here and there, and know that metal knockers were being
broken, but knowing also that they might as well try to catch a will-o’-the-
wisp as one of the perpetrators.
It must be admitted that playing such practical jokes as these is poor fun,
and the only thing to be said for the students is, that they never paused to
think.
. . . . . .
Sandie still stuck to his little pupil, though he confessed more than once
to Willie that the work was irksome in the extreme.
Our hero was no gourmand. And yet there were many Highland students
at the University, who lived on far poorer fare than did Sandie, as we shall
see as the story goes on.
Sandie had porridge and milk for breakfast, nothing else, but plenty of
that. For dinner he usually had sheep’s-head broth of barley and vegetables,
with potatoes and perhaps kail as auxiliaries. He allowed himself tea in the
afternoon, and for supper a large dish of stiff pease-meal brose with plenty
of creamy milk.
When fresh herrings could be got—but they were not now in season—he
treated himself to a few of these.
This was plain, but it was also wholesome fare.
Herring and sand cadgers are quite a feature of the Granite City. What
the poor people do with all the fine sea-sand it would be difficult to
imagine. But the Aberdonians are a cleanly people, the very show of their
white granite walls appears to suggest cleanliness, and the women folks are
constantly seen scouring down their stairs and passages.
The sand is hawked in donkey-carts, and the boy hawkers’ are invariably
all in rags and tatters.
“Twa buckets o’ fine sea-sand for three bawbees, and I’ll carry them
upstairs for a cauld tattie or a bit o’ cake for the cuddy.”
I may state at once that the cuddy never gets the piece of cake.
The herring-cadgers are a cut above the sand-laddies.
In going to classes one day shortly before Christmas, Sandie was witness
to a rather humorous episode. Let me premise that the streets were covered
with mud and slime.
Well, a large cart-load of hay from the country had just met and passed a
cadger’s cart laden with fresh herrings. This was an excellent opportunity to
get a wisp of hay, thought the herring-man, so he was speedily helping
himself to an armful. But Geordie spied him, and off he went to the cart and
quite filled his arms with herrings.
“Faur (where) are ye gaun wi’ my herrin’?” cried the cadger aghast.
“Faur are ye gaun wi’ my hay?” answered Geordie.
“There’s you dirty hay,” shouted the cadger, throwing it on the ground.
“And there’s your dirty herrings,” cried Geordie, throwing the fish in the
mud, which certainly would not improve either their flavour or quality.
But Geordie had the best of it.

CHAPTER V

AMONG THE WHITE HARES—HOGMANAY NIGHT

When Christmas-time came round, Sandie M‘Crae not only felt that he
needed a week’s rest, but that he had worked hard enough to deserve one. It
was therefore with a feeling of intense enjoyment and pleasure that he
seated himself in the train, his merry little friend Willie by his side, the train
that should soon bear him far away to his own bonnie Highland home and
his ain fireside.
Oh, that ain fireside, which nought surrounds save an atmosphere of
love, how pleasant it is to think of when far, far away! Sandie had thought
of it often and often when hard at work in his little attic, and longed to be
there. The loving father, seated in his arm-chair, quietly smoking; the
gentle-faced mother, bending over her knitting; his sweet sister Elsie, with a
book; the cat and the bawsent-faced collie Tyro.
Quickly enough sped the train, but under the circumstances it is no
wonder Sandie thought it slow. His head is out through the window long
before he nears the station. Yes, he can see Elsie with the dogcart and Lord
Raglan, and he waves his handkerchief to her, and she smilingly waves her
hand in return, for Elsie and Sandie are all in all to each other.
Sandie is in such a hurry that he almost forgets to give up his ticket. He
rushes off the little platform, and next moment is almost capsized by Tyro
himself, who is perfectly wild in his demonstrations of joy and undying
love.
“Oh,” he seems to tell Sandie, “I thought I would never, never see you
more; I thought you were dead and away, and now, what can I do to allay
my feelings?”
And in order to do so the poor dog must commence flying round and
round in a circle, so quickly that his shape is barely distinguishable. Having
fondly embraced his sister, and asked after his father and mother, and while
Willie and she are shaking hands, Sandie takes Raglan’s head in his arms to
cuddle. Then he kisses his soft snout, and the horse whinnies a welcome.
Sandie next takes a paper parcel from his ulster and opens it, extracting
therefrom great slabs of white oat-cake.
“Lord Raglan,” he cries, “I didn’t forget you.”
Raglan whinnies once more, and probably enjoys that cake far more than
he has enjoyed anything for many a day.
Tyro also has a share. Then all wheel happily home to the farm of old
Kilbuie.
 . . . . . .
“I shan’t touch a classic or open a book on mathematics until we return
to college.” That is what Sandie told Willie next morning at breakfast.
“Well, now, I do call that wise,” replied Willie; “one doesn’t expect
much wisdom from a genius—one doesn’t really, but for once in a way
——”
“Thank you,” said Sandie.
“And you’ll eat all you can, laddie,” quoth Sandie’s mother, “and drink
plenty o’ milk, for indeed you’re as white as a ghost.”
“Mother dear,” replied Sandie, “I’ll do all you tell me, even to the
drinking of milk, and right glad I am to have the chance of obeying you
once again.”
“O mother and Siss,” he added, with something akin to exultation, “I
used often and often to dream about this good old-fashioned fireside, and
then waken all alone in my attic so cold and dismal!”
. . . . . .
Of course, one of the first visits the boys made was to the manse of
Belhaven.
The first person they saw was Maggie May herself. She ran joyously to
meet Sandie, holding out both her hands. But she did not present her face to
be kissed.
“I do declare, Maggie May,” said our hero, “you appear to have grown
since I saw you last.”
“Yes,” said the girl, “I suppose I must have.” Then she blushed bonniely
as she added, “You must remember I am quite old now, thirteen last
birthday.”
“And you’ve had a birthday since I’ve been here, and I was not aware of
it! How hard is fate! Never mind, Maggie May, I’ve brought you something
for a Christmas present. Oh, I shan’t keep you guessing what it is, and you
shall have it now. I have it here.”
Sandie went to the little dogcart and produced a box, and Maggie May’s
eyes sparkled as she opened it and took therefrom a charming and well-
filled cartridge-belt.
Of course she tried it on at once, and it fitted her nicely, and became her
very much.
 “And my little gift,” said Willie, presenting a little box. It contained a
pair of beautiful earrings, that Maggie May thought must have cost a small
fortune, so studded with precious stones were they.
About this moment Maggie May was probably the happiest girl in the
parish.
Presently Mackenzie himself came in, then conversation became
general.
“What think you of the weather?” said Sandie at last.
“I’ll tell you what it is,” answered the minister, “before twenty-four
hours are over there will be a slight fall of snow and no wind.”
“Hurrah!” cried Sandie.
“I know why you cry ‘Hurrah!’ You’re thinking about the white hares!”
“That’s it.”
“Well, it is all arranged. There will be you, Sandie, Maggie May here,
my man Stuart, and my simple self.”
“And me,” added Willie, with small regard for grammar.
“Well, now, as a friend,” said Sandie, “I’m going to be very
straightforward. You remember your last sporting venture, and the
somewhat original way you loaded your gun? Well, I think that this time
you had better stay at home, Willie, and talk to my mother and Elsie.”
“I’ve got a new gun,” said Willie doggedly. “You’ve only to put in a
cartridge and hold it out, and she goes off beautifully.”
“Yes; and perhaps shoots your neighbour.”
“Sandie M‘Crae, first Bursar of Marischal College, I’m going. That is
decided.”
Sandie sighed.
“A wilful man must have his way,” he said.
The white or mountain hare, reader, is found plentifully in Norway and
among our Scottish hills. It is not white all summer, but only changes to that
colour when winter comes, a kind of provision of nature to hide it from its
enemies, the fox and the eagle, and probably the great owl.
Its life is a hard one among the frozen hills in winter. Oftentimes its poor
paws will be found skinned and bleeding from scratching the hard ground
in order to procure a little food. They are usually stalked when snow is on
the ground, their footsteps being followed, so that dogs are not really
necessary.
But on this expedition, undertaken by our hero and his friends, the
minister’s retriever, Carlo, made one of the party.
It may be thought wonderful that a mere child like Maggie May should
be permitted to join a venture like this across the bleak and frozen hills.
Sandie had suggested her staying at home.
“She’s a true Mackenzie,” said the minister. “A Mackenzie is nothing at
all if not hardy. Believe me, Maggie will keep up with the best of us.”
Stuart, who had no gun, carried the luncheon and looked after the dog.
The trap was left at a little croft not far from a high steep hill, and then
the party proceeded on foot.
There was broom to struggle through at first, then heather to wade
among, so high that it nearly buried Maggie May. Sandie stuck by her side,
helping her in every difficulty.
But as they reached higher ground, the heather grew shorter, and ere
long entirely disappeared; then, to their great joy, they came upon the
footprints of apparently several hares.
Cautiously they followed them up for some distance. Suddenly Willie
brought his gun to the shoulder.
Bang! Carlo bounded forwards and returned next minute with a splendid
specimen of the mountain hare.
“Good, Willie, good!” cried Sandie, grasping him by the hand. “But
wasn’t it a——”
“A fluke? I think not.”
No one there had such quick eyes as Willie, for in five minutes more he
repeated his first exploit, and a short time afterwards he did the same again.
As Mackenzie and the others looked so thoroughly and completely
astonished, Willie was forced to laugh aloud.
“Oh, you humbug!” cried Sandie. “Why you’re a crack shot, you rascal,
and that episode of loading the gun was got up to deceive us!”
“Look! look!” This from Willie, as two splendid ptarmigans rose from
the ground.
 Mackenzie and Willie fired a barrel each, and both birds fluttered
groundwards.
Well might Willie smile. He had established his fame as a good shot, and
completely wiped out the stain from his character as a sportsman.
On and on all that forenoon went the party, no one seeming to feel the
least tired. But towards two o’clock they began to feel hungry, if they did
not feel tired, for the air among these Highland hills is keen and bracing. So
Stuart spread plaids on the snow, and down they all sat to one of the most
delightful luncheons ever partaken of by hungry huntsmen. It was now
nearly three o’clock, and the winter’s sun was rapidly nearing the pine
forest on the rugged shores south of the Don. So all haste was made back to
the trap, Sandie assisting Stuart in carrying the hares and birds. As they
mounted the trap to drive back to the manse, everybody agreed that they
had spent a glorious day.
Willie, nevertheless, confessed to being tired.
“Well,” said Sandie, “we’ll forgive you for that; but, O Willie, what a
trick you played us!”
But the refreshing cups of tea that Maggie May brought Willie, when at
last they got safely home, banished every vestige of fatigue, and he was
soon his laughing, happy self again.
As the wind had now begun to blow, and snow was falling, the students
agreed to stay at the manse all night. So a messenger was despatched
immediately to Kilbuie farm to let Mrs. M‘Crae know their decision, and
then Mackenzie, who really was a boy at heart, and the students settled
down to enjoy themselves.
The minister rather prided himself on the good dinners he gave, and
certainly that of to-night was no exception to the general rule.
After this, as Robbie Burns says—

“The nicht drave on wi’ sangs and clatter,”

till twelve o’clock, chimed out by the pretty clock on the mantelpiece, a gift
from the minister’s parishioners, warned them it was time to court repose.
. . . . . .
How quickly that week sped away, only those situated as were Sandie
and Willie could imagine.
 But every time has an end, and the more we are enjoying ourselves, the
faster does old Father Time fly. This is very nasty of old Father Time, only
he will have his own way, despite anything we can say or do.
The last night had come and gone, and Willie had retired to his room,
and was seated by the window, through which the bright moonlight was
streaming, when Elsie, looking in her long night-dress like a sheeted ghost,
came gliding in. Her dark hair all undone was streaming down her back.
Sandie hastened to place a seat for her, and to wrap her from top to toe in a
Highland plaid.
All in all were they to each other that brother and sister, and innumerable
were the things they had to tell each other on this last night, and many the
confidences to interchange, for four long months must elapse ere they could
see each other again.
More than once Sandie could see tears glistening in the moonbeams on
his sister’s cheeks.
But one o’clock came at last, and he had to send her away.
“Anyhow, Sandie,” she said, as she rose to go, “you will promise not to
study too, too hard. Mind you are all I have, Sandie, and if anything
happened to you, the grave would soon close over your poor sister Elsie.”
“I promise,” said Sandie, “to take care of myself for mother’s sake and
yours. Good-night, dear Elsie.”
“Good-night, dear Sandie.”
And away glided the girl again as silently as she had come.
. . . . . .
Sandie and Willie got back to the city on Hogmanay night. That is the
last night of the old year. This is kept in Scotland with great glee, and I fear
with not a little drunkenness. No one thinks of going to bed till the New
Year comes in, and no one thinks of remaining indoors.
Our heroes found Union Street about eleven o’clock crowded to excess,
one dense mob from Union Bridge to Castle Hill, but all good-humoured,
all hearty. Here and there the bagpipes skirled, here and there songs were
sung.
But when it was within about five minutes to twelve an expectant hush
fell over all that vast multitude.
 Anon the first stroke of the bell boomed over the city, then the cheer that
went toward that moonlit sky may be imagined, but never never could be
described.
At the same moment everybody seemed to produce a bottle of whisky,
and everybody drank with and shook hands with his nearest neighbour, no
matter who or what he was.
But by one o’clock the multitude had melted away, solitary watchmen
paraded the streets, and the pale moon shown calmly down on the pure
white walls of the Granite City.

CHAPTER VI

IN SNOW-TIME—A TOWN AND GOWN

My well-beloved reader—what a pretty expression, by the way!—must not
jump to the conclusion that this chapter, and those that follow, describe life
at the Northern University far back in the Middle Ages.
No; Sandie’s time was just about thirty years ago. Ten years after that, I
know there was but little change. There may or may not be an alteration
since, for I have been to sea, and scarcely clapped eyes on a red gown.
Well, in Sandie’s time, town and gown riots were far from uncommon;
especially in snow-time. Snow-time was glow-time then. The very look of
the falling snow sent a thrill of joy to each Grammar School boy’s or even
student’s heart, and the first question one would ask another would be—
“Is it making?”
That is, was the snow soft enough to form easily into snowballs? For if
very frosty and powdery it was of course no use. As most of the real
snowball battles took place just when the thaw commenced, a constant
fusillade would then be carried on all up and down Union Street. The street
boys, as well as students, were chokeful of mischief, and every conspicuous
person caught it hot—if a snowball can be called hot. Battered silk hats
were scattered in all directions. Mashers or extra-well-dressed people
became simply living targets; silk umbrellas, if put up, were speedily
riddled—it was only a case of making the snowball a trifle harder, an extra
squeeze did that, and lo! there was a hole in the silken ’brella.
 It is almost needless to say that the bobbies, or policemen, suffered
greatly at such times. In fact, a policeman was hardly to be seen without an
expanded snowball or two on his greatcoat, and more than one might be
sporting black eyes. As for catching the depredators, and running them in,
this was out of the question. The running-in part would have been easy
enough, but first they had to catch their hare,—there was the rub.
Well, school challenged school. The Grammar School, for example,
dominated the Gordons, or Sillerton boys, with a rod of iron.
These boys, in those days, were the drollest-looking chaps it is possible
to conceive. They used to march four deep, with a bit of a fife and drum
band ahead of them; and, just imagine it, they were all dressed like little old
men, in blue swallow-tailed coats, with brass buttons, knee-breeches, and
broad Tam o’ Shanter bonnets.
Well, on days when the snow was making, the Grammar School lads
would lie in wait for them, about three deep on each side of the street, and
when they got the Gordons right between, oh, then the fun began, and soon
waxed fast and furious. Some of the teachers, foolishly enough, would
charge the Grammarians with their umbrellas. They were soon to be pitied;
here and there you would see one of these well-dressed whiskered dons
lying on his back, his umbrella torn to tatters, and snowballs alighting on
his person from all directions, as if from a Maxim gun.
Meanwhile the Gordon ranks would be broken up, the music stopped,
and after perhaps an ineffectual attempt at self-defence, Sillerton would be
demoralised and flying for safety in all directions.
But there were other schools that would meet the Grammar School at
times. I have known them meet by challenge by the Denburn side, and a
fine afternoon’s fight be the result.
Then there used to be a manufactory where the workers were terrible
roughs, namely, “the comb-work chaps,” as they were called. As a rule, the
Grammar School steered clear of these. They were bad to beat, and there
was no honour or glory in beating them. Besides, they used to put stones
inside their snowballs.
Sometimes bands of sailor boys used to come up from the shipping in
the harbour to engage the Grammar School in a pitched battle, and all up
and down the school-hill the fight would rage sometimes for hours.
 Once I remember the Grammar School was being badly beaten by the
comb-work chaps. Many had received ugly cuts in their faces with stoned
snowballs.
The school lads were almost demoralised, and making a running fight of
it towards their own quad. But help was at hand. A band of red gowns had
heard of the brutality of those roughs, and now they managed to outflank
the cowardly ruffians, while the Grammar School boys rallied once again
and attacked them from the front.
Desperate diseases require desperate cures, and in this case the students
despised snowballs. Those cads used stones, let them have it. This was the
cry, and the red gowns went at them tooth and nail, or stick and fist. It
ended by the comb-work chaps receiving such a drubbing that they were
civil for all the season thereafter. They seemed determined now not again to
provoke a fight with the Grammar School boys, who had such fierce and
terrible allies in those wild hordes of red gowns.
“Where were the policemen?” it may be asked, when fights like these
were going on. I think I would be safe to say they were somewhere round
the corner. One dutiful bobbie might go to his sergeant, and a conversation
such as the following would take place:—
Bobbie. “Man! sairgent, there’s an unco killo-shangie (riot) goin on at
the tap o’ Jack’s brae!”
Sergeant. “Ye dinna say so? What’s doin?”
B. “Oh, Grammarians, comb-work chaps, and students—they’re a’ at it.”
S. “Ony (any) windows broken?”
B. “I canna say there is.”
S. “Weel, man, just lat them fecht awa. They canna hurt ane-anither
(each other); a black e’e or a bloody nose’ll do them good, and we canna
help it. Laddies will be laddies.”
B. “A’ richt then. I’ll keep oot o’ sicht.”
S. “Ay, do.”
. . . . . .
“Now, Sandie,” cried Willie, one morning in the end of January, as he
burst gleefully into his friend’s attic and surprised him at his porridge, “I’ve
good news for you. You and I are both invited to the medical students’
supper, the night after next.”