Mammalian Species Origin and Geographical Dispersal Patterns

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5-2012 Mammalian Species Origin and Geographical Dispersal Patterns Correlate With Changes in Chromosome Structure, Exemplified in Lemurs (Madagascar) and Bats (Worldwide) Robin Lee Kolnicki University of Massachusetts Amherst, [email protected]

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MAMMALIAN SPECIES ORIGIN AND GEOGRAPHICAL DISPERSAL PATTERNS CORRELATE WITH CHANGES IN CHROMOSOME STRUCTURE, EXEMPLIFIED IN LEMURS (MADAGASCAR) AND BATS (WORLDWIDE)

A Dissertation Presented

ROBIN LEE KOLNICKI

Submitted to the Graduate School of the

University of Massachusetts Amherst in partial fulfillment

of the requirements for the degree of

DOCTOR OF PHILOSOPHY

May 2012

Geosciences

MAMMALIAN SPECIES ORIGIN AND GEOGRAPHICAL DISPERSAL PATTERNS CORRELATE WITH CHANGES IN CHROMOSOME STRUCTURE, EXEMPLIFIED IN LEMURS (MADAGASCAR) AND BATS (WORLDWIDE)

A Dissertation Presented

ROBIN L. KOLNICKI

Approved as to style and content by:

______

Michael F. Dolan, Chair

______

Richard W. Wilkie, Member

______

Elizabeth R. Dumont, Member

______

Jeffrey Podos, Member

______

R. Mark Leckie, Department Head Geosciences

DEDICATION

With tremendous respect and love, to Lynn Margulis, who is the motivation behind this pursuit of understanding speciation.

ACKNOWLEDGMENTS

I thank Neil Todd for insight into the recognition of patterns of chromosome fission found in each mammal family; Lynn Margulis for recognizing the significance of the kinetochore (i.e. an ubiquitous eukaryotic microtubular organizing center found on chromosomes), reproductive behavior in evolution; and Richard Wilkie for inspiring the search for correlation between evolutionary innovation and geographical location.

ABSTRACT

MAMMALIAN SPECIES ORIGIN AND GEOGRAPHICAL DISPERSAL PATTERNS CORRELATE WITH

CHANGES IN CHROMOSOME STRUCTURE, EXEMPLIFIED IN LEMURS (MADAGASCAR) AND BATS

(WORLDWIDE)

MAY 2012

ROBIN LEE KOLNICKI, B.A., AMERICAN INTERNATIONAL COLLEGE

M.Ed., AMERICAN INTERNATIONAL COLLEGE

M.S., UNIVERSITY OF MASSACHUSETTS AMHERST

Ph.D., UNIVERSITY OF MASSACHUSETTS AMHERST

Directed by: Professors Michael Dolan and Lynn Margulis

The origin and geographical distribution of mammalian species (my examples are lemurs and bats) correlate with predictable chromosomal structural changes (KFT=karyotypic fission theory). Chromosome studies provide information about fertility between individuals and they are significant for identification of the geographical origin of reproductive isolation within mammal families. Each family predictably has chromosome sets with numbers that range from one to double the lowest number of chromosomes. The chromosome numbers of all species within a single family are used to reconstruct that family’s evolutionary geographical dispersion.

Polymorphic chromosome numbers (that is a range such as 34, 35, 36, 37, and 38) in a single population indicate the location where chromosomal diversification arose. Chromosome numbers of descending order correlate with relative distance from fission epicenters as the fissioned chromosomes gradually spread to neighboring populations. Furthermore, the location of chromosomal diversification (that is “karyotypic fission events) is associated with

geographical “zones of transition” (after Professor R.W. Wilkie). My analysis, mapped one

(Lepilemuridae) of the five families of lemurs (Class Mammalia, Order Primates, sub-order

Lemuridae). The origin of this family’s diversification is here hypothesized to have occurred at an ecological transition zone in Northern Madagascar between a humid evergreen-forest that extends to the East relative to a dry deciduous forest along the West Coast. My analysis of

Vespertilionidae (insectivorous bats representing one third of all bat species) suggests a diversification event occurred in Asia; South China.

Geographical distribution is important in the formation of biological diversity. A single species can inhabit a wide range and exhibit great diversity that is brought about by natural selection. The Holarctic reindeer found in Scandinavia, Russia, China, Canada and Alaska

(including caribou) are all a single species Rangifertarandus that exhibits variation in size and in coat pattern, changes brought about by adaptive selection by the environment or human selective breeding but they all have 70 similar chromosomes and they are all reproductively compatible. There is a single species of reindeer. Although, there is measurable DNA sequence divergence; there has been no “speciation” as these circumpolar cervids are genetically compatible.

vii

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... v ABSTRACT ...... vi LIST OF TABLES ...... ix LIST OF FIGURES ...... x

CHAPTER

1. KINETOCHORE REPRODUCTION IN ANIMAL EVOLUTION ...... 1

2. KARYOTYPIC FISSION THEORY APPLIED, KINETOCHORE REPRODUCTION AND LEMUR EVOLUTION ...... 8

3. CHROMOSOME CHANGE IN THE EVOLUTION OF LEMUR SPECIES ...... 28

4. LEMURS AND SPLIT CHROMOSOMES ...... 67

5. CHROMOSOMAL CHANGES, ZOOGEOGRAPHICAL DISPERSAL AND MAMMALIAN SPECIES ORIGIN ...... 84

6. RANGIFER TARANDUS RUMEN MICROBIAL SYMBIONTS PERMIT HOMO SAPIENS SURVIVAL IN EXTREME ARCTIC ENVIRONMENT ...... 105

GENERAL BIBLIOGRAPHY ...... 132

viii

LIST OF TABLES

Table Page

2.1 Lemur karyotype references ...... 12

3.1 Primary literature on lemur karyotypes ...... 49

6.1 The symbiotic microorganisms required for digestion of food in the rumen of the free lving reindeer Rangifer tarandus ...... 120

6.2 Seasonal variation in ciliate (protist) and bacteria populations within the reindeer rumen and cecum microbial communities ...... 122

LIST OF FIGURES

Figure Page

1.1 (A) Dicentric chromatid segregation. (B) Chromatids replicate, and fertilization leads to offspring heterozygotic for the dicentric chromomosome ...... 4

2.1. Fusion and fission theory compared for lemur evolution ...... 14

2.2. Homologous chromosomal combinations ...... 15

2.3. Nonviable hybrid combinations ...... 16

2.4. Modern diploid ranges constrain postulated prefissed ancestral chromosome arrangements ...... 17

2.5 Karyological phylogeny of lemurs...... 18

2.6. Lemurid karyological evolution ...... 19

2.7. Chromosomal evolution of Eulemur macaco (2N=44) ...... 22

3.1. Karyological phylogeny of lemurs ...... 53

3.2. Lepilemurid karyological evolution ...... 55

3.3. Lemurid and cheirogaleid karyological evolution ...... 57

3.4 Chromosomal evolution of cheirogaleids and lemurids ...... 59

3.5. Lepilemurid karyological evolution ...... 60

4.1 A phylogeny of the five lemur families ...... 81

4.2. Karyotypic fission generates four functional chromosomes ...... 82

5.1. Biogeographical distribution of Lepilemur chromosome numbers ...... 100

6.1. The rumen symbio-organ of reindeer (Rangifera tarandus) harbors a community of symbiotic micro-organisms that are essential for the digestion of plants, lichens, and mushrooms ...... 120

CHAPTER 1

KINETOCHORE REPRODUCTION IN ANIMAL EVOLUTION: CELL BIOLOGICAL EXPLANATION OF

KARYOTYPIC FISSION THEORY

Kolnicki, R.L. 2000. Kinetochore reproduction in animal evolution: Cell biological explanation of karyotypic fission theory. Proceedings of the National Academy of Sciences 97: 9493-9497.

1.1 Introduction

1.2 Karyotype Evolution in Animals

1.3 Synchronous Reproduction of Kinetochores

1.4 Seeding New Kinetochores

1.5 Kinetochore Protein Dephosphorylation Regulates Chromosome Segregation

1.6 Double-Strand Breakage Between Kinetochore Pairs

Figure 1.1: (A) Dicentric chromatid segregation. (B) Chromatids replicate, and fertilization leads to offspring heterozygotic for the dicentric chromomosome.

1.7 De Novo Telomeres

1.8 Synapsis and Segregation of Acrocentric Derivatives

1.9 Pericentric Inversions

1.10 Conclusions

1.11 References

CHAPTER 2

KARYOTYPIC FISSION THEORY APPLIED: KINETOCHORE REPRODUCTION AND LEMUR

EVOLUTION

Kolnicki, R.L. 1999. Karyotypic Fission Theory Applied: Kinetochore Reprodcution and Lemur Evolution. Symbiosis, 26:123-141.

2.1 Abstract

2.2 Introduction

2.3 Karytopic Fissioning and the Evoluton of Lemurs

Table 2.1: Lemur karyotype references (continued onto next page)

Figure 2.1: Fusion and fission theory compared for lemur evolution

Figure 2.2: Homologous chromosomal combinations

2.4 Pericentric Inversions

Figure 2.3: Nonviable hybrid combinations 16

2.5 Ancestral Chromosomal Arrangements

Figure 2.4: Modern diploid ranges constrain postulated prefissed ancestral chromosome arrangements. 17

Figure 2.5: Karological phylogeny of lemurs.

Figure 2.6: Lemurid karyological evolution.

2.6 Lemur Phylogeny

2.7 Lemurdae Phylogeny

2.8 A Mechanism for Karyotypic Fissioning

Figure 2.7: Chromosomal evolution of Eulemur macaco (2N=44)

2.9 Conclusions

2.10 Acknowledgements

2.11 References

CHAPTER 3

CHROMOSOME CHANGE IN THE EVOLUTION OF LEMUR SPECIES

3.1. Abstract

Karyotypic fission theory applied to the mammalian order Primates, suborder Strepsirrhini, explains the diversity of chromosome numbers (diploid numbers from 20 to 70) and their morphology in all five lemur families (lepilemurids, daubentoniid, lemurids, cheirogaleids and indriids). Kinetochore reproduction provides the cell-level mechanism for lemur evolution via karyotypic fission as inferred from karyological analysis. Modern lemurs evolved from an ancestral primate with a diploid number of 20 mediocentric chromosomes. Lepilemuridae karyotypes (2N= 20 to 38) evolved from the ancestral 2N = 20 by a single primary karyotypic fission event. Daubentoniidae, with only one species (2N=30), resulted from a single independent fission event. Lepilemurids and daubentoniids are therefore the least derived, karyologically, from a basal lemur ancestor. Lemurid and cheirogaleid karyotypes (2N= 40 to 62, and 2N= 46 and 66) by contrast, result from a shared secondary fission event. Indriid karyotypes

(2N=40 to 48, and 70) evolved from the basal ancestor by a secondary fission independent of the lemurid/cheirogaleids. Varying retention of ancestral mediocentric linkages and centromere relocation phenomena fully generate modern karyotypic morphology of all lemurs.

Key words: centromere, Cheirogaleus, chromosome, evolution, Indri, Lepilemur, neocentromere,

Primate

3.2. Introduction

Karyotypic fission theory applied to lemurs explains the evolutionary history of all five families.

Details for the Lemuridae were published (Kolnicki, 1999), here the theory is applied to the remaining four families: Lepilemuridae, Daubentoniidae, Cheirogaleidae, and Indriidae.

Kinetochore reproduction theory (Kolnicki, 2000) provides the molecular/cellular basis for lemur chromosomal diversification by fission.

3.2.1. Karyotypic Fission

Karyotypic fission theory was developed by Todd, (1967) to explain the relation of chromosome number and morphology to "adaptive radiations," that is, species diversification as inferred from both anatomy and the fossil record of mammals. He posited that the ancestral chromosomal complement for all mammals is comprised of fourteen large mediocentric (i.e., metacentric, submetacentric, or subtelocentric) chromosomes from which karyotypic fission events generated karyotypes with higher diploid numbers and smaller chromosomes. Such episodes of chromosome fission introduce a full complement of smaller homologous acrocentric derivatives in populations with mediocentric chromosomes. Varying retention of ancestral mediocentric chromosomes and incorporation of fission-generated acrocentric pairs potentially generates a diploid range that has up to twice the ancestral number of chromosomes. The retention of the extant ancestral non-fissioned sex chromosomes appears, he suggested, to be favored by selection. The accumulation of random mutations generally is assumed to underlie reproductive

isolation and speciation. Todd's fission model, a view more consistent with extant artiodactyl, carnivore and old world monkey and ape karyotypes, correlates adaptive radiations with dramatic karyotypic changes that effect linkage relationships. Banding studies show all marsupial karyotypes are derived from a conserved 2N=14 karyotype (Rofe and Hayman, 1985).

Comparative chromosome painting, fluorescence in situ DNA hybridization, supports the hypothesis that 2N=14 is ancestral in marsupials (Toder, et al., 1998, DeLeo et al., 1999). Imae's

(2001) mathematical model of chromosomal evolution based on minimal interaction theory depicts diverse animal and insect karyotypes as best explained by fission "bursts" followed occasionally by single fusions. Fission "bursts" apparently underlie species diversification. They minimize deleterious reciprocal translocations associated with long chromosome arms. The large range of mammalian diploid numbers (2N = 6 to 92) is most parsimoniously explained by

Todd’s karyotypic fission theory.

Early on, Todd's fission theory, originally based on karyotype analysis coupled with zoogeographic and paleontological information, was ignored or received little support because no mechanism for fission was thought to exist (Godfrey and Masters, 2000). Chromosome mutations were taken to occur randomly and one at a time by "centromeric breaking"; most or all of the kinetochore/centromere was thought to remain with only one of the resulting acrocentrics. The consequence was assumed to be improper meiotic segregation and aneuploidy (White, 1973). But the new work in biochemical cytology, that led to kinetochore reproduction theory and provided the cellular/molecular processes for the synthesis of supernumerary kinetochore/centromeres, (Kolnicki, 2000) revived awareness of Todd's original concept. 30

The assumption that the ancestral karyotype for all primates was 20 large mediocentric chromosomes is consitant with all data on extant animals. Three karyotypic fission events fully explain karyotype evolution in old world monkeys and apes: a primary event during the late

Eocene produced the karyotypic diversity in the Catarrhini (2N=42 to 48); a secondary event during the Miocene produced the karyotypes in the Pongidae and Hominidae (2N=46 to 48), and another secondary event that occurred some time after 20 million years ago at the beginning of the Miocene epoch produced the high diploid numbers (ranging from 2N=54-72) in the genus

Cercopithecus (Guisto and Margulis, 1980). Chromosome painting studies that use fluorescence in situ DNA hybridization support the concept that primate karyotypes evolve by chromosome fission. Chromosome painting of African green monkey (2N= 60) and human (2N = 46) chromosomes with outgroup analysis indicates an evolutionary history of increasing diploid numbers through fission (Finelli et al., 1999). In the evolution of the galago prosimian primates, comparative chromosome painting indicates the significant role played by fission (Stanyon et al.,

2002). Here, I apply karyotypic fission theory to lemurs (2N=20 to 70) and show that four fission events, two primary and two secondary, in principle could have generated all their karyotypes.

Homology between metacentric chromosomes (in one species, race, or individual) and two smaller acrocentrics in another was first noted by Robertson (1916). "Robertsonian rearrangement" refers thus to both chromosome fission and fusion. This karyotypic theory requires all mediocentric chromosomes fission to yield two smaller fully homologous acrocentrics in a single event that involves the kinetochore/centromeres*. Fusion, by contrast,

occurs independently for each chromosome with no necessary involvement of the kinetochore/centromere. Two smaller chromatin fragments join to form a larger chromosome.

Robertsonian rearrangements are relatively common throughout animal taxa. They contribute to both interspecific and intraspecific karyological polymorphism. Fission-generated chromosomes are favored by selection during meiotic chromosome segregation. Analysis of

1170 mammalian karyotypes heterozygous for Robertsonian translocations shows chromosomes with supernumerary kinetochore/centromeres "attach preferentially to the pole that is most efficient at capturing centromeres" (Pardo-Manuel de Villena and Sapienza, 2001).

Nonrandom segregation of chromosomes during meiosis in the female drives karyotypic evolution towards an increase in kinetochore/centromere number. The Robertsonian chromosomal variation reported for numerous animal taxa is most parsimoniously explained by karyotypic fission theory where kinetochore/centromere reproduction provides the cell-level mechanism that underlies the phenomena (reviewed in Kolnicki, 2000 and discussed below).

* The terms kinetochore and centromere both refer to the attachment site of chromosomes to the mitotic spindle. Until the mid20th century these names were considered synonymous (Wilson, 1925). Analysis of the structures required for segregation of condensed chromatin (the chromosomes are primarily composed of DNA and histone protein) to the poles of the cell indicate that all “kinetochore-centromeres” namely, spindle fiber attachments, are composed of both DNA (“centromeric DNA”) and proteins (“kinetochore proteins”). The several kinetochore proteins include “motors,” proteins necessary for movement of the kinetochore-centromeres along the spindle microtubules to the cell poles. For this reason, I use here the term kinetochore/centromere to refer to the functioning structure. In its absence DNA replicates but is not segregated to the offspring cells. “Neocentromere” refers to the relocation of the kinetochore/centromere on a chromosome. Neocentromere emergence can change acrocentric chromosomes to metacentrics and vice versa.

3.2.2. Lemur Taxonomy

Lemurs, strepsirrhine primates indigenous to Madagascar, represent approximately one-third of all living primate species. Combined morphological and DNA data analysis suggest Malagasy primates originated from a single common ancestral population that diverged from other primates by the early Eocene approximately 50 million years ago, at the latest (Yoder, 1996). Of the eight lemur families, five (Lepilemuridae, Daubentoniidae, Lemuridae, Cheirogaleidae, and

Indriidae) are extant (Jenkins, 1987). The taxonomy of lemurs in this analysis follows

Mittermeier et al. (1994) except that Lepilemur is not included in the extinct Megaladapidae.

Instead, following Jenkins (1987), Lepilemur is given separate familial status.

The Lepilemuridae, "sportive lemurs" and “weasel lemurs,” comprises at least six recognized species: Lepilemur ruficaudatus, L. edwardsi, L. dorsalis, L. leucopus, L. mustelinus, and L.

septentrionalis. The Northern sportive lemur, L septentrionalis, is karyologically polymorphic

(2N=34, 35, 36, 37, and 38). Four subspecies; L. septentrionalisseptentrionalis, L. septentrionalis ankaranensis, L. septentrionalis sahafarensis, and L. septentrionalis andrafiamensis were suggested on the basis of their distinct karyotypes (Rumpler and Albignac, 1975). The revision of L. septentrionalis taxonomy to include two chromosomally polymorphic species based on cytogenetic analysis of 60 karyotypes was proposed by Rumpler et al. (2001).

The Lemuridae includes ten recognized species; Eulemur macaco, E. coronatus, E. rubriventer, E. fulvus, E. mongoz, Lemur catta, Hapalemur griseus, H. aureus, H. simus, and Varecia variegata.

Taxonomic status of some Eulemur species remains contentious because of “intraspecific” fertility variability and natural hybridization that occurs between members of separate

"species." Six morphologically distinct "subspecies" that have five distinguishable karyotypes

(2N=48, 50, 51, 52, and 60) have been assigned to the brown lemur, Eulemur fulvus, found widely on Madagascar. Intraspecific crosses between “subspecies” E. f. collaris (2N=52) and E. f. albocollaris (2N=48) show variability in the degree of fertility in offspring (Tattersall, 1993). The separation of these animal groups into two species based on reduction of fertility linked to chromosomal multivalent formation during meiosis was suggested by Djlelati, et al (1977). That natural interspecific hybridization exists between E. f. rufus and E. mongoz (mongoose lemur) populations (both 2N=60; Zaramody, 2001) indicates further taxonomic revision may be necessary. The diversity of Eulemur karyotypes are best explained as the result of a single karyotypic fission event (Kolnicki, 1999).

The Cheirogaleidae includes at least nine species: Microcebus murinus, M. myoxinus, M. ravelobensis, M. rufus, Mirza coquereli, Allocebus trichotis, Cheirogaleus medius, C.major, and

Phaner furcifer. Microcebus,"mouse lemurs," are a diverse group of the World’s smallest living primates. A taxonomic revision of Microcebus species based on morphological and molecular

(i.e. mtDNA sequence, COII and cytochrome b) data was suggested by Yoder (2000). Three new species: M. berthae (the smallest primate species), M. sambiranensis, and M. tavaratra are described and M. myoxinus and M. griseorufus are resurrected from synonymy.

Mirza,Cheirogaleus and Allocebus are slightly larger "dwarf lemurs." Cheirogaleus major consists of five species, and C. medius of two based on the morphological characters of museum specimens (Groves, 2000). Phaner furcifer, “the fork-marked lemur,” has been assigned four subspecies distinguished by both morphology and geographical range (Groves and Tattersall,

1991).

Only a single extant species, Daubentonia madagascariensis, the aye-aye, is placed in the family

Daubentoniidae. Its unique dental formula, ever-growing incisors, large ears, and long middle finger used for extracting insect larvae from wooddistinguishes Daubentonia from all other lemurs. Because of Daubentonia’s distinctive autapomorphies, its evolutionary relationship to the other lemurs has long been controversial. Recent chromosome painting studies supports an early separation of Daubentoniidae from all other lemurs (Warter, 2005)

Three genera and six species: Avahi laniger, A. occidentalis, lndri indri, Propithecus verreauxi, P. diadema, and P. tattersalli are assigned to the Indriidae family. "Woolly lemurs,” the Avahi

species are small nocturnal primates. Propithecus, "sifakas," are a morphologically diverse group of larger diurnal lemurs. Indri, that weighs 6 to 7 kg., and is easily distinguished from

Propithicus species by it’s vestigial tail, is the largest living lemur (Mittermeier, 1994).

3.3. Materials and Methods

3.3.1. Lemur Karyotypes

The karyotypes used in this analysis and their sources are listed in Table I. Conflicting earlier karyological data for certain species (e.g., Lemur catta, Eulemur fulvus fulvus, Eulemur mongoz,

Lepilemur leucopus, Lepilemur dorsalis, Propithecus diadema, and Avahi laniger) has been superceded by newer reports used here. Advances in cytological protocol and use of DNA staining (Geimsa, R-banding, Q-banding, and fluorescence in situ hybridization) have improved the accuracy of karyotype evaluation.

3.3.2. Ancestral Diploids

Ancestral diploid numbers are inferred from reconstruction of the ancestral morphotypes that would, by karyotypic fission, yield the modern karyotypes. The lowest diploid number found in primates is 20. Lepilemur ruficaudatus has 20 large mediocentric chromosomes and is thus hypothesized here to closely approximate the ancestral condition for all lemurs. A primary karyotypic fission event yields the diploid number range of 20 to 38 found in lepilemurids.

Secondary fission events are postulated to generate diploid numbers higher than 38. The range of diploid numbers within each family provides constraints for postulating basal chromosomal arrangements. Chromosome morphology (i.e. chromosome size, kinetochore/centromere

location, DNA banding and fluorescence markers), provides further constraint on the ancestral condition that best explains each family's karyotypes.

3.3.3. Kinetochore/Centromere Relocation

Each modern karyotype is compared to the postulated fission-generated chromosomal arrangement that has the same diploid number to determine the likelihood of kinetochore/centromere relocation and the directionality of the shift. The original karyotypic fission theory posited that subsequent to fission the acrocentrics that convert to mediocentric chromosomes repotentiate secondary fission (Todd, 1967). Todd’s concept that fission- generated acrocentrics converted to mediocentric chromosomes is bolstered by modern study of the relocation of the kinetochore/centromeres via neocentromere formation or pericentric inversion (Kolnicki, 2000). Newly available DNA banding and chromosome painting studies help determine if kinetochore/centromeric rearrangement is indeed shared between species. I assume here that unidirectional changes in kinetochore/centromere location (i.e., metacentric to acrocentric or vice versa) found among closely related species were acquired prior to species separation for parsimony unless published reports indicate otherwise. That any species incorporated a chromosome rearrangement independently from another is determined by banding or marker analysis.

3.4. Results

3.4.1. Ancestral primate diploid number of 20

If I postulate that all lemurs evolved from an ancestral primate (2N=20) the karyotypes within five families can be generated by four fission events (Fig. 3.1). A primary karyotypic fission event in the ancestral lemuriform population would have generated the postulated chromosome diploid numbers of the basal daubentoniids, lemurids, cheirogaleids and indriids.

Subsequent rearrangement, particularly kinetochore/centromere relocation, theoretically isolates Daubentoniidae and Indriidae from each other and from other lemur families. A separate primary karyotypic fission event in the basal lepilemurids (2N=20) generates the diverse arrangements found in Lepilemuridae (2N=20 to 38). A shared secondary fission event in a polymorphic population (2N=34) that varies by one chromosome pair (acrocentric or mediocentric) fully explains modern Lemuridae (2N= 44 to 62) and Cheirogaleidae (2N=46, and

66) karyotypes. A separate secondary fission event in a population with a mediocentric chromosomal arrangement (2N=38) explains indriid karyotypes (2N=40 to 48, and 70). With no recourse to other chromosome mutation fission alone accounts for chromosome numbers and karyotype morphology (i.e., the number of mediocentric and acrocentric chromosomes) exhibited by many species of lemurs (particularly in the genera Eulemur, Varecia, Lemur,

Cheirogaleus, Hapalemur, Microcebus, and Allocebus).

3.4.2. Lepilemurid Karyotype Analysis

Based on karyotypic fission theory all modern Lepilemurid karyotypes (2N=20, 22, 26, 34, 35, 36,

37, and 38) evolved directly from a primary fission event from the postulated ancestor (2N=20)

(Fig. 3.2). The hypothetical lemuriform ancestral chromosomal condition is closely approximated in Lepilemur ruficaudatus. L. edwardsi, L. mustelinus and L. septentrionalis subspecies incorporated a single kinetochore/centromere relocation that converted a pair of

acrocentric autosomes to mediocentrics. A single shared kinetochore/centromere shift from which L. ruficaudatus populations were excluded occurred prior to the divergence of lepilemurid species. Kinetochore/centromere relocation accounts for the acrocentric X chromosome found in L. edwardsi. Kinetochore/centromere relocation on two or three autosome pairs fully explains the reported karyotypes of L. dorsalis and L. leucopus (2N=26). L. dorsalis and L. leucopus were reported to have 18 mediocentric and 6 acrocentric autosomes (Rumpler and

Albignac, 1978, Rumpler, 1975). More recent reports by the same investigator (Rumpler et al.

1985, Rumpler et al. 1986) indicate their karyotypes have 20 mediocentric and 4 acrocentric autosomes. Since the arrangements reported earlier are not addressed in more recent papers, it is questionable whether these species are truly polymorphic for kinetochore/centromere location or if the earlier interpretation of autosomal morphology is inaccurate. Recent evidence that kinetochore/centromere relocation is prevalent in primates justifies the inclusion here of both karyotypes. L. septentrionalis subspecies have polymorphic diploid numbers: L. septentrionalis septentrionalis (34), L. septentrionalis ankaranensis (36), L. septentrionalis sahafarensis (36),and L. septentrionalis andrafiamenensis (38).The highest lepilemurid diploid number, in L. septentrionalis andrafiamenensis (2N=38), is the highest theoretically derivable from a fission event in an ancestral population with a diploid number of 20 given the retention of the non-fissioned X.

3.4.3. Daubentoniid Karyotypes

Daubentonia madagascariensis also evolved from the ancestral lemurid (2N=20). The large size of the 24 metacentric and 4 acrocentric autosomes indicates that no secondary fission occurred.

The primary fission event generated an arrangement with 8 mediocentric and 20 acrocentric

autosomes. The modern (2N=30) karyotype of Daubentonia is reconciled by hypothesizing kinetochore/centromere relocation on eight autosome pairs.

3.4.4. Lemurid and Cheirogaleid Karyotypes

All Lemuridae and Cheirogaleidae karyotype morphologies were generated by a single secondary karyotypic fission event in a polymorphic population (3N=34) where one autosome pair was acrocentric or mediocentric, respectively. A phylogeny for lemurid and cheirogaleid evolution based on karyotypic fission theory is illustrated in Figure 3.3. The basal karyotypes

(2N=34) arose from a primary karyotypic fission event followed by kinetochore/centromere relocation on acrocentric derivatives, by hypothesis. Modern lemurid karyotypes (2N=44, 46,

48, 50, 51, 52, 54, 56, 58, 60, and 62) result from the secondary fission event. No other chromosomal rearrangement needs to be hypothesized with the exception of Hapalemur aureus

(2N=62) which incorporated kinetochore/centromere relocation on a single autosome pair. All cheirogaleids except Phaner furcifer have similar karyotypes (2N=66) directly explained by secondary fission. Cheirogaleus major and C. medius are claimed to have a karyotype (2N=66) with 2 mediocentric and 62 acrocentric autosomes (Wurster-Hill, 1973). If Cheirogaleus were truly polymorphic, a single shared kinetochore/centromere relocation would explain this chromosome morphology. Phaner (2N=46) likely incorporated two kinetochore/centromere shifts. These data support the hypothesis that lemurid and cheirogaleid karyotypes are derived from a single chromosomally polymorphic population with a diploid number of 34.

My hypothetical evolutionary transition from the ancestral lemuriform (2N=20) to the modern karyotypes of Cheirogaleus, Microcebus, Allocebus (all 2N=66) and Eulemur mongoz (2N=60) is

diagrammed in Figure 3.4. The modern lemur most similar to the postulated ancestral lemur karyotype is Lepilemur ruficaudatus whose chromosomes vary in size. I illustrated the ancestral chromosome arrangement for all lemurs with uniform chromosome size only to simplify the representation. A primary fission event in the postulated lemuriform ancestral condition (Fig.

3.4A) yields (2N=34) with 4 mediocentric and 28 acrocentric autosomes (Fig. 3.4B).

Kinetochore/centromere relocation converted acrocentric to mediocentric autosomes with no effect on the diploid number (Figs. 3.4C, 3.4E). A fission event in this karyologically polymorphic basal population generated all of the Lemuridae and Cheirogaleidae chromosomal diversity.

Cheirogaleus, Microcebus and Allocebus karyotypes are comprised of small acrocentric autosomes (Fig. 3.4D). The four largest pairs of acrocentric autosomes evolved from a single fission event whereas the other autosomes were produced by secondary fission. The karyotype of Eulemur mongoz (2N=60) is explained by retention of one large pair of ancestral mediocentric linkages, three pairs of medium-sized acrocentric autosomes (one from the primary and two from the secondary fission events), one pair of smaller mediocentric autosomes (from the primary fission event and subsequent kinetochore/centromere relocation), and finally twenty- four pairs of very small acrocentric autosomes from the secondary fission event (Fig. 3.4F). That all lemurids retain one large pair of ancestral mediocentric autosomes and one medium sized pair of acrocentric autosomes from the primary fission event is consistent with the modern karyotypes.

3.4.5. Indriid Karyotypes

The indriid karyotype phylogeny (Fig. 3.5) is based on a secondary karyotypic fission event that generated modern indriid karyotypes with (0, 1, or 2) kinetochore/centromere relocations. Indri

indri, with the lowest diploid number (2N=40) underwent one or possibly two autosomal kinetochore/centromere relocations. The actual morphology of small chromosomes is in question. The Indriidae challenge karyotypic analysis in that the morphology of their numerous small chromosomes is determined with difficulty. Three distinct karyotypes (2N=40, 42, and 44) have been reported for Propithecus diadema subspecies. In 1973, Rumpler published a report that the karyotype of P. diadema diadema (2N=42) has 32 metacentric and 8 acrocentric autosomes. Rumpler later reports that the smallest pair of chromosomes found in P. diadema and P. verreaxi is "difficult to interpret" with respect to morphology; appearing "in many preparations as acrocentric and in others as metacentric" (Rumpler and Albignac 1980). In

1988, Rumpler questions the validity of his earlier interpretations in that the actual chromosome number may have been misinterpreted because of the difficulty in viewing microchromosomes. Four distinct karyotypes have been published for Avahi laniger (2N=64, 66, and 70). The diploid number of 64 tentatively reported (Rumpler, 1975) based on an analysis of a single individual was never mentioned subsequently. The diploid number of 66 is in two reports (Petter, Albignac, and Rumpler 1977; Rumpler and Albignac 1980). The karyotype

(2N=66) suggested in 1980 is based on a single metaphase spread. A more recent evaluation

(Rumpler et al. 1990) reports that there are only two karyotypic arrangements present in Avahi and both have a diploid number of 70 but vary in chromosome morphology. Only the 1990 report (2N=70) is included in this analysis. The karyotypes of lndri, Propithecus diadema diadema and P. diadema perrieri are probably not polymorphic. P. d. diadema and P. d. perrieri

(both 2N=42) karyotypes evolved directly from the postulated fission event with no more than one kinetochore/centromere relocation. The karyotype of Propithecus diadema edwardsi

(2N=44) was generated by kinetochore/centromere relocation on one autosome pair. The

Propithecus verreauxi karyotype (2N=48) was generated by kinetochore/centromere relocation on one or two autosome pairs. Avahi laniger apparently incorporated a single kinetochore/centromere shift, an observation consistent with published DNA banding analysis

(Rumpler et al., 1990).

3.5. Discussion

3.5.1. Ancestral condition

Lepilemur ruficaudatus, with its 20 large mediocentric chromosomes closely approximates the ancestral condition for all lemurs. DNA banding pattern analysis (R-banding, C-banding, and Ag-

NOR staining) of lepilemurid chromosomes supports a hypothesized ancestral diploid number of twenty (Rumpler, et al., 1986). Fission and kinetochore/centromeric relocation are the most frequent rearrangements in lemur chromosomal evolution as shown by comparative chromosome banding analysis of Lemuridae (Eulemur fulvus), Cheirogaleidae (Microcebus murinus) and Indriidae (Avahi laniger and Propithecus verreauxi) karyotypes (Rumpler et al.,

1983, Warter, 2005). Four karyotypic fission events generate the diverse chromosomal morphologies found in lemurs.

Although the scenario here posits the most parsimonious reconstruction of lemuriform karyotypic evolution and karyotype analysis provides useful constraints on drawing phylogenetic inferences other phylogenies are not necessarily excluded. The recent analysis by Warter et al.

(2005) indicates an early separation of Daubentoniidae. Early separation of

Daubentoniidaesuggests an additional fission event isolated it from the other families. My

postulation that Daubentonia’s chromosomal complement underwent fission is consistent with

Warter’s (2005) interpretation that Daubentonia’s karyotype differs from all other lemurs by eight rearrangements. A primary fission event followed by kinetochore/centromere relocation on eight autosomes generates Daubentonia’s modern karyotype.

The view that the basal cheirogaleid and basal lemurid share a common trunk is consistent with recent cytogenetic studies. Comparative chromosome painting by zoo-FISH indicates a unique

Robertsonian translocation is shared by cheirogaleids and lemurids (Warter, 2005). That the basal cheirogaleid and basal lemurid karyotypes (both 2N=34) differed by kinetochore/centromere location on a single autosome pair is consistent with DNA banding pattern analysis. The cheirogaleid karyotype of Microcebus murinus differs by one pericentric inversion from karyotypes found in lemurids (Rumpler and Dutrillaux, 1976).

3.5.2. Karyotypic Fission

I conclude that the prevalence of polymorphic karyotypes in lemurs is most parsimoniously explained by karyotypic fission theory. Both cytological evidence and hybrid studies show that

Robertsonian rearrangements predominate chromosomal evolution in lemurs. The intraspecific chromosomal variation found in Lepilemur septentrionalis (2N=34, 36, or 38) is determined by inheritance of zero, one, or two Robertsonian rearrangements (Rumpler, 1975). Naturally occurring hybrids of L. septentrionalis (2N=35, 37) are heteromorphic for one chromosome pair; they have one mediocentric chromosome paired with two smaller acrocentric homologs

(Rumpler, 1975). Eulemur fulvus collaris has a polytypic arrangement (2N=50, 51, 52; Hamilton,

1977). E. f. collaris (2N=51), heteromorphic for a Robertsonian rearrangement on one

chromosome pair, has a mediocentric chromosome paired with two smaller acrocentric homologs. Crosses between E. f. collaris (2N=51) and E. f. rufus (2N=60) show homologous pairing of 5 metacentric with 10 acrocentric autosomes verified by DNA banding of chromosomes (Moses, 1979). A natural hybrid zone exists between populations of E. f. albocollaris (2N=48) and E. f. rufus (2N=60) (Wyner, 2002). Interspecific crosses between E. f. fulvus (2N=60) and Eulemur macaco (2N=44) produce both fertile and sterile hybrids (Tattersall

1993; Dutrillaux and Rumpler 1977; Albignac et al. 1971). Crosses between E. fulvus fulvus

(2N=60) and E. rubriventer (2N=50) produce viable but sterile hybrids (Saint-Pie 1970). Analysis of the hybrid chromosome arrangement shows 10 acrocentric autosomes in E. fulvus fulvus pair with 5 submetacentrics in E. rubriventer (Rumpler and Dutrillaux, 1980). Hapalemur griseus

“subspecies” are distinguished by Robertsonian rearrangement (2N=54, 55, 56, and 58; Rumpler et al., 2002). Four metacentric chromosomes in Phaner (2N=46) correspond to eight acrocentric chromosomes in Microcebus murinus (2N=66) (Rumpler and Dutrillaux 1979). The key result is that whereas over 100 independent Robertsonian rearrangements would be required to explain modern lemur karyotypes, only four are needed to explain lemur karyotype evolution by karyotypic fission theory.

3.5.3. Sex Chromosomes

The metacentric X chromosome comprising approximately 5% of the genome is a relatively constant feature in mammalian karyotypes, a fact that led Todd to assume that karyotypic fission events do not alter the sex chromosomes. However, lack of uniformity with respect to the X chromosome in lemurid karyotypes requires examination of this assumption. L. edwardsi is the only lepilemurid with an acrocentric X sex chromosome. Banding pattern analysis suggests a pericentric inversion (Rumpler and Dutrillaux, 1990). All Lemuridae species have 45

acrocentric sex chromosomes with the exception of: Varecia variegata, Hapalemur simus and

Lemur catta. Earlier reports based on DNA banding studies suggest pericentric inversion to explain lemurid sex chromosomes (Rumpler and Dutrillaux 1978, 1979). Analysis of chromosome marker order in lemurid sex chromosomes indicates kinetochore/centromere relocation via neocentromere emergence (Ventura M, et al., 2001). The formation of neocentromeres and their subsequent stabilization are consistent with the karyotypic fission view with respect to kinetochore reproduction. I posit that acrocentric X chromosomes in lemurids were not derived by fission but rather through synthesis of new kinetochores.

3.5.4. Kinetochore/Centromere Relocation

Common to all indriids are centromeric shifts or "change in the position of the kinetochores"

(Rumpler et al., 1990). Avahi laniger and A. occidentalis share a similar diploid chromosome number (2N=70) but differ in their chromosome morphology by a single pericentric inversion unique to Avahi laniger as shown by DNA banding (Rumpler et al., 1990). Of interest, lndri indri has a secondary constriction on the long arm of chromosome 3 near the kinetochore/centromere (Rumpler and Albignac, 1980). Kinetochore/centromere relocation via neocentromere formation, followed by chromosome fission or inactivation of one kinetochore, is consistent with the karyotypic fission view of chromosomal evolution.

3.5.5. Fission-generated Chromosomes

Synthesis of supernumerary kinetochores on meiotic chromatids generates dicentric chromosomes. Subsequent chromosome breakage between the centromere pairs yields smaller acrocentrics complete with functional kinetochores as diagramed in Fig. 3.1. The enzyme,

telomerase, usually found in high levels in gametes, accounts for de novo telomere sequences that cap broken chromosome ends and preclude fusion or translocation. Telomerase activity thus stabilizes fission-generated chromosomes. A single plausible event: kinetochore/centromere reproduction during gametogenesis affects all chromosomes. This chromosomal rearrangement would lead to entirely normal offspring with no significant alteration of gene sequence, total DNA quantity, or other phenotypic change. Subsequent chromosomal rearrangement, especially kinetochore/centromere relocation or pericentric inversion, would increase the probability of genetic isolation amongst incipient sympatric species polytypic for fission-generated acrocentric chromosomes as Todd first postulated.

Fissioned karyotypes would repotentiate for secondary fission events after small acrocentric chromosomes convert to mediocentric (Todd, 1967). Difficulty in pairing a single acrocentric autosome, generated by primary fission, with a smaller homologous acrocentric pair (generated at the secondary fission event) theoretically creates a genetic barrier; i.e., reproductive isolation between members in the population with incompatible chromosomal morphology (Fig. 3;

Kolnicki, 1999). Kinetochore reproduction theory attributes crucial significance to changes in kinetochore/centromere reproduction that, coupled with eventual reproductive isolation, lead to species diversification. So far karyotypic fission theory has been applied to Carnivora (Todd,

1970), Artiodactyla (Todd, 1975, 2000), and Primates (Guisto and Margulis, 1980), but equines and bats are underway and many more taxa are amenable to analysis.

Kinetochore behavior plays a significant role in chromosomal evolution. Kinetochores, composed of proteins located at centromeric DNA, bind microtubules to form spindle and are the motors of chromatid segregation during karyokinesis (mitotic nuclear division). Centromere

DNA replication is temporally separate from that of other chromatin. The evolutionary rate of change in centromere heterochromatic DNA sequences is more rapid than in euchromatin. In primates, centromeric and pericentromeric sequences have a high degree of plasticity when compared with euchromatic domains (Jackson et al., 1999). Centromere chromatin is distinguished from euchromatin by its specialized nucleosome structure (Blower et al., 2002), distinct level of coiling (Bloom et al., 1989), and by its unique histone (H3) composition (Malik,

2001). Mutations in centromere DNA are useful in determination of evolutionary relatedness of species, races, or populations. Primate kinetochore/centromere position exhibits an evolutionary history independent from surrounding chromosome markers (Montefalcone, et al.,

1999). Chromosome painting studies indicate a prevalence of epigenetic kinetochore formation for which neocentromere emergence provides the best explanation. A minimum common centromeric structural DNA motif is probably required for "epigenetic" assemblage of kinetochores (Abad and Villasante, 2000). Taken together these data establish karyotypic fission and kinetochore/centromeric relocation as the major factor in speciation of lemurs. The generation of approximately 50 distinct taxa in 14 genera of Lemuridae in five families in the last

65 million years of evolution on the island of Madagascar coupled with recent research is best attributable to Todd’s 35-year old theory.

Table 3.1: Primary literature on lemur karyotypes (continued onto next two pages)

Lemur Species 2N M A XY Reference

Lepilemur ruficaudatus 20 18 0 MA Rumpler, Ishak, Warter and Dutrillaux 1985.

Lepilemur edwardsi 22 18 2 AA Rumpler, Ishak, Dutrillaux, Warter and Ratsirarson 1986.

Lepilemur dorsalis 26 18 6 MA Rumpler 1975.

26 20 4 MA Rumpler, Ishak, Dutrillaux, Warter and Ratsirarson 1986.

Lepilemur leucopus 26 18 6 MA Rumpler and Albignac 1978.

26 20 4 MA Rumpler, Ishak, Warter and Dutrillaux 1985.

Lepilemur mustelinus 34 6 26 SA Rumpler, Ishak, Dutrillaux, Warter and Ratsirarson 1986.

Lepilemur 34 6 26 MA Rumpler, Ishak, Warter and Dutrillaux septentrionalis 1985. septentrionalis

L. septentrionalis 35 5 28 MA Rumpler, and Albignac 1975.

L.s. ankaranensis 36 4 30 MA Rumpler, Ishak, Warter and Dutrillaux 1985.

L.s. sahafarensis 36 4 30 MA Rumpler and Albignac 1975.

L. septentrionalis 37 3 32 MA Rumpler and Albignac 1975.

L.s. andrafiamenensis 38 2 34 MA Rumpler and Albignac 1975.

Daubentonia 30 24 4 MA Tagle, Goodman and Miller 1990. madagascariensis

Eulemur macaco 44 20 22 AA Rumpler and Dutrillaux 1976.

Eulemur coronatus 46 18 26 AA Rumpler and Dutrillaux 1979.

Eulemur fulvus fulvus 48 16 30 AA Chu and Swomley 1961.

60 4 54 AA Rumpler and Dutrillaux 1990.

E f. albocollaris 48 16 30 AA Rumpler and Dutrillaux 1976.

E f. collaris 50 14 34 AA Hamilton, Beuttner-Janusch and Chu 1977.

51 13 36 AA Hamilton, Beuttner-Janusch and Chu 1977.

52 12 38 AA Rumpler and Dutrillaux 1976.

E. f. rufus 60 4 54 AA Chu and Swomley 1961.

E f. albifrons 60 4 54 AA Rumpler and Dutrillaux 1980.

E f. sanfordi 60 4 54 AA Rumpler 1975.

Eulemur mongoz 60 4 54 AA Rumpler and Dutrillaux 1990.

Eulemur rubriventer 50 14 34 AA Rumpler and Dutrillaux 1980.

Varecia variegata 46 18 26 SA Rumpler and Dutrillaux 1979.

Lemur catta 56 8 46 MA Rumpler and Dutrillaux 1978.

Hapalemur griseus 54 10 42 AA Rumpler and Albignac 1973.

8 44 AA Chu 1975.

H. g. alaotrensis 54 10 42 AA Rumpler 1975.

H. g. meridionalis 54 10 42 AA Warter, Randrianasolo, Dutrillaux and Rumpler 1987.

H. g. spp. 56 8 46 AA Rumpler and Dutrillaux 1978.

H. g. occidentalis 58 6 50 AA Rumpler and Dutrillaux 1978.

H.g. olivareus 58 6 50 AA Chu 1975.

Hapalemur simus 60 4 54 SA Rumpler, Prosper, Hauwy, Rabarivola, Rakotoarisoa and Dutrillaux 2002.

Hapalemur aureus 62 0 60 AA Rumpler, Warter, Hauwy, Randrianasolo and Dutrillaux 1991.

Microcebus murinus 66 0 64 MA Rumpler and Dutrillaux 1976. murinus

M. m. rufus 66 0 64 MA Rumpler and Albignac, R. 1973.

Mirza coquereli 66 0 64 SA Rumpler and Dutrillaux 1979.

Allocebus trichotis 66 0 64 MA Rumpler, Warter, Hauwy, Meier, Peyrieras, Albignac,Petter and Dutrillaux 1995.

Cheirogaleus major 66 2 62 MA Wurster-Hill 1973.

0 64 SA Rumpler and Dutrillaux 1979.

Cheirogaleus medius 66 2 62 MA Wurster-Hill 1973.

0 64 MA Rumpler and Albignac 1973.

Phaner furcifer 46 16 28 MA Rumpler and Dutrillaux 1979.

Avahi occidentalis 70 4 64 SA Rumpler, Warter, Rabarivola, Petter and Dutrillaux 1990.

Avahi laniger 70 6 62 SA Rumpler, Couturier, Warter and Dutrillaux 1983.

66 4 60 SA Petter, Albignac and Rumpler 1977.

Indri indri * 40 30 8 SA Rumpler, Warter, Ishak and Dutrillaux 1988.

32 6 MA Rumpler and Albignac 1973.

Propithecus diadema 42 30 10 MA Rumpler and Albignac 1980 diadema * 32 8 MA Rumpler and Albignac 1973.

Propithecus diadema 42 32 8 MA Rumpler and Albignac 1973. perrieri 40 32 6 -- Rumpler and Albignac 1973.

Propithecus diadema 44 32 10 SA Rumpler, Warter, Ishak and Dutrillaux edwardsi 1988.

Propithecus diadema 48 30 16 SA Rumpler, Couturier, Warter and Dutrillaux 1983. verreauxi *

28 18 -- Rumpler and Albignac 1980.

M = Mediocentric (metacentric and submetacentric), A = Acrocentric and S = Submetacentric chromosomes, * = there is difficulty in determining the actual morphology of smallest autosomes.

Figure 3.1: Karyological phylogeny of lemurs.

Lemur karyotypes are most parsimoniously explained by karyotypic fission (KF) followed by kinetochore/centromere relocation (K-CR). The common ancestral Lemuriform karyotype

(2N=20), all mediocentric autosomes, is maintained in the Lepilemuridae. All modern lepilemurid karyotypes result from a single KF. An earlier, independent, KF yields polymorphic diploid numbers, three of which (2N=30, 34 and 38) generate the karyotypes of all extant species in the Daubentoniidae, Lemuridae, Indriidae, and Cheirogaleidae. The karyotype of

Duabentonia madagascarensis is explained the primary KF and subsequent K-CR. Indriid karyotypes are explained by KF in a chromosomal arrangement (2N=38) having all mediocentric autosomes. KF in a population with karyological polymorphism (2N=34) having 30 mediocentric and 2 acrocentric autosomes or 32 mediocentric and 0 acrocentric autosomes generates the karyotypes of Lemuridae and Cheirogaleidae. A single autosome pair is illustrated in to show morphological changes that result from fission, subsequent kinetochore/centromere relocation, and secondary fission. Each pair of large mediocentric chromosomes potentially yields four pairs of smaller homologous acrocentric chromosomes. (Modified from Kolnicki, 2000)

Figure 3.2: Lepilemurid karyological evolution.

Lepilemur ruficaudatus (2N=20) with all mediocentric autosomes closely approximates the hypothetical ancestral lemuriform population. Karyotypic fission and kinetochore/centromere relocation on one chromosome that spread through the post fissioned population generated polymorphic karyotypes (2N=22, 26, 34, 35, 36, 37, 38). Kinetochore/centromere relocation on two or three additional autosomes (2N=26) explains the L. dorsalis and L. leucopus karyotypes.

Karyotype morphology is represented as 2N = M (mediocentric autosomes) + A (acrocentric autosomes) + XY (sex chromosomes). Shaded boxes represent centromere relocation; AM = acrocentric is converted to mediocentric chromosome via neocentromere activation or pericentric inversion as discussed in the text. Since two separate karyotypes were published for

L. dorsalis and L. leucopus (Rumpler and Albignac 1978; Rumpler et al. 1985; Rumpler et al.

1986) and that they are polytypic is not established with certainty, both karyotypes are considered here.

Figure 3.3: Lemurid and cheirogaleid karyological evolution.

A shared secondary karyotypic fission event explains all modern chromosomal arrangements.

Theoretically, the basal lemurid/cheirogaleid population differed in karyotype morphology by a single acrocentric or metacentric autosome pair, respectively. One secondary karyotypic fission

(KF) event yields modern diploid numbers and chromosome morphology.

Kinetochore/centromere relocation that converted a mediocentric autosome pair to acrocentric 57

(MA) accounts for the karyotype of Hapalemur aureus. Kinetochore/centromere relocation on two autosomes (MA) generates the modern karyotype of Phaner furcifer.

Figure 3.4: Chromosomal evolution of cheirogaleids and lemurids.

A) By hypothesis the ancestral lemur autosomal chromosomes were all mediocentrics (2N=20).

B) A karyotypic fission event in this population of ancestors generated a karyotype (2N=34). C,

E) Kinetochore/centromere relocation converted acrocentric autosomes to mediocentrics with no change in diploid number. D, F) Cheirogaleid and lemurid karyotypes result directly from a shared secondary fission event.

Figure 3.5: Indriid karyological evolution.

Key: KF = karyotypic fission, AM = acrocentric to mediocentric kinetochore/centromere relocation, MA = mediocentric to acrocentric kinetochore/centromere relocation, karyotypes are represented as 2N = M (mediocentric autosomes) + A (acrocentric autosomes) + XY (sex chromosomes).

3.6 References

Abad, J. P. and Villasante, A. 2000. Searching for a common centromeric structural motif: Drosophila centromeric satellite DNAs show propensity to form telomeric-like unusual DNA structures. Genetica 109:71–75.

Albignac, R., Rumpler, Y. and Petter, J. J. 1971.L’hybridation des lemuriens de Madagascar. Mammalia 35:358–368.

Bloom, K., Hill, A., Kenna, M. and Saunders, M. 1989.The structure of a primitive kinetochore.Trends Biochem.Sci. 14, 233–227.

Blower M. D., Sullivan B. A. and Karpen, G.,H. 2002. Conserved organization of centromeric chromatin in flies and humans. Dev. Cell. 2:319–330.

Chu, E. H. Y. 1975. Hapalemur griseus. In: An atlas of mammalian chromosomes. Hsu, T. C. and Binirscheke, Kl, ed., 9:441.

Chu, E. H. Y. and Swomley, B. A. 1961.Chromosomes of lemurine lemurs. Science 133:1925– 1926.

DeLeo, A. A., Guedelha, N., Toder, R., Voullaire, L., Ferguson-Smith, M. A., O’Brien, P.C. and Graves, J.A. 1999. Comparative chromosome painting between marsupial orders: relationships with a 2N = 14 ancestral marsupial karyotype. Chromosome Res. 7:509–517.

Djlelati, R., Brun, B. and Rumpler, Y. 1997. Meiotic study of hybrids in the genus Eulemur and taxonomic considerations. Am. J. Primatol. 42:235–245.

Dutrillaux, B., Rumpler, Y. 1977. Chromosomal evolution in Malagasy lemurs. II. Meiosis in intra- and interspecific hybrids in the genus Lemur. Cytogenet.Cell.Genet. 18:197–211.

Finelli, P., Stanyon, R., Plesker, R., Ferguson-Smith, M. A., O'Brien, P. C. and Wienberg, J. 1999. Reciprocal chromosome painting shows that the great difference in diploid number between human and African green monkey is mostly due to non-Robertsonian fissions. Mamm.Genome. 10:713–718.

Godfrey, L.R. and J. Masters. 2000. Kinetochore reproduction theory may explain rapid chromosome evolution. Proceedings of the National Academy of Sciences.97:9821–9823.

Groves, C.P. 2000. The genus Cheirogaleus: Unrecognized biodiversity in dwarf lemurs. Int. J. Primatol. 21: 943–962.

Groves, C.P. and I. Tattersall. 1991. Geographical variation in the fork-marked lemur Phaner furcifer (Primates, Cheirogaleidae). Folia.Primatol. 56:39–49.

Guisto J. and L. Margulis. 1980. Karyotypic fission theory and the evolution of Old world monkeys and apes. BioSystems 13:267–302.

Hamilton, A. E., Beuttner-Janusch, J. and Chu, E. H. 1977.Chromosomes of lemuriformes. II. Chromosome polymorphism in Lemur fulvus collaris (E. Geoffroy 1812). Am. J. Phys. Anthropol. 46:395–406.

Imai HT, Satta Y, Takahata N. 2001. Integrative study on chromosome evolution of mammals, ants and wasps based on the minimum interaction theory. J Theor Biol Jun 21;210(4):475-97.

Jackson, M. S., Rocchi, M., Thomson, G., Hearn, T., Crozier, M., Guy, J., Kirk, D., Mulligan, L., Ricco, A., Marzella, R., Viggiano, L., and Archidiacono, N. 1999. Sequences flanking the centromere of human chromosome 10 are a complex patchwork of arm-specific sequences, stable duplications, and unstable sequences with homologies to telomeric and other centromeric locations. Hum. Mol. Genet. 8:205–215.

Jenkins, P. D. 1987. Catalogue of primates in the British museum (Natural History) and elsewhere in the British Isles. Part IV: Suborder Strepsirrhini, including the subfossil Madagascan lemurs and family Tarsiidae. British Museum (Natural History), London.

Kolnicki, R.L. 1999. Karyotypic fission theory applied, kinetochore reproduction and lemur evolution. Symbiosis 26:123–141.

Kolnicki, R. L 2000. Kinetochore reproduction in animal evolution: Cell biological explanation of karyotypic fission theory. Proceedings of the National Academy of Sciences.97:9493–9497.

Malik, H.S. and Henikoff, S. 2001. Adaptive evolution of cid, a centromere-specific histone in drosophila. Genetics 157:1293–1298.

Mittermeier, R. A., Tattersol, I., Konstant, W. R., Meyers D. M. and Mast R.B. 1994.Lemurs of Madagascar, Conservation International Tropical Field Guide Series, Conservation International, Washington, DC.p.128.

Montefalcone, G., Tempesta, S., Rocchi, M. and Archidiacono, M. 1999.Centromere repositioning. Genome Res. 9:1184–1188.

Moses, M.J., Karatsis, P. and Hamilton, A. 1979.Synaptonemal complex analysis of heteromorphic trivalents in lemur hybrids. Chromosoma 70: 141–160.

Pardo-Manuel de Villena, F. and Sapienza, C. 2001. Female meiosis drives karyotypic evolution in mammals. Genetics 159:1179–1189.

Petter, J.J., Albignac, R. and Rumpler, Y. 1977.Faune de Madagascar 44: Mammiferes Lemuriens (Primates Prosimiens) O.R.S.T.O.M./C.N.R.S., Paris, 474–482.

Rofe, R. and Hayman, D. L. 1985. G-banding evidence for a conserved complement in the Marsupialia.Cytogenet.Cell Genet. 39:40–50.

Robertson, W. R. B. 1916. Chromosome studies I. Taxonomic relationships shown in the chromosomes of Tettidgidae and Acridididae. V-shaped chromosomes and their significance in Acrididae, Locustidae and Gryllidae: chromosomes and variation. J. Morphol. 27:179–331.

Rumpler, Y. 1975. Chromosomal studies in systematics of Malagasy lemurs. In: Lemur Biology, Tattersol, I. and Sussman, R., eds. Plenum Press, New York. Pp. 25–40.

Rumpler, Y. and Albignac, R. 1973. Cytogenic study of the Intrudae Burnett 1828, a malagasy endemic Lemur’s family (International Congress of Anthropological and Ethnological Sciences– conference and workshop on comparative kariology of primates), Detroit and Chicago.

Rumpler, Y. and Albignac, R. 1975. Intraspecific chromosome variability in a lemur from the north of Madagascar: Lepilemur septentrionalis, species nova. Am. J. Phys. Anthropol. 42:425– 429.

Rumpler, Y. and Albignac, R. 1978. Chromosome studies of the Lepilemur, and endemic Malagasy genus of lemurs: contribution of the cytogenetics to their taxonomy. J. Hum. Evol. 7:191–196.

Rumpler, Y. and Albignac, R. 1980.Cytogenetic study of the Indridae Bennet, 1832, an endemic Malagasy lemur family. In B. Chiarelli, A.L. Koen, and G. Ardito, eds.: Comparative Karyology of Primates (Mouton Publishers, New York).

Rumpler, Y. and Dutrillaux, B. 1976.Chromosomal evolution in Malagasy lemurs III.Chromosome banding studies in the genuses Lemur and Microcebus.Cytogenet.Cell Genet. 17:268–281.

Rumpler, Y. and Dutrillaux, B. 1978.Chromosomal evolution in Malagasy lemurs I. Chromosome banding studies in the genus Hapalemur and the species Lemur catta. Cytogenet.Cell.Genet. 21:201–211.

Rumpler, Y. and Dutrillaux, B. 1979.Chromosomal evolution in Malagasy lemurs IV. Chromosome banding studies in the genuses Phaner, Varecia,Lemur, Microcebus, and Cheirogaleus. Cytogenet.Cell Genet. 24: 224–232.

Rumpler, Y. and Dutrillaux, B. 1980. Chromosomal evolution in Malagasy lemurs V. Chromosomal banding studies of Lemur fulvus albifrons, Lemur rubriventer and its hybrids with Lemur fulvus fulvus. Folia primatol. 33:253–261.

Rumpler, Y. and Dutrillaux, B. 1990.Chromosomal evolution and speciation in primates.Cell Biol. Rev. 23:1–136.

Rumpler, Y., Couturier J, Warter S. and Dutrillaux B. 1983.Chromosomal evolution in Malagasy lemurs VII.Phylogenetic relationships between Propithecus, Avahi (Indridae), Microcebus (Cheirogaleidae), and Lemur (Lemuridae).Cytogenet.Cell Genet. 36:542–546.

Rumpler, Y., Ishak, B., Warter, S. and Dutrillaux, B. 1985.Chromosomal evolution in Malagasy lemurs VIII. Chromosome banding studies of Lepilemur ruficaudatus, L. leucopus, and L. septentrionalis. Cytogenet.Cell Genet. 39:194–199.

Rumpler, Y., Ishak, B, Dutrillaux, B., Warter, S. and Ratsirarson, J. 1986.Chromosomal evolution in Malagasy lemurs. IX. Chromosomal banding studies of Lepilemur mustelinus, L. dorsalis, and L. edwardsi. Cytogenet.Cell Genet. 42:164–168.

Rumpler, Y., Warter, S., Ishak, B. and Dutrillaux, B. 1988. Chromosomal evolution in Malagasy lemurs: X. Chromosomal banding studies of Propithecus diadema edwardsi and Indri indri and phylogenic relationships between all the species of the Indriidae. Am. J. Primatol. 16: 63–71.

Rumpler, Y., Warter, S., Rabarivola, C., Petter, J.J. and Dutrillaux, B. 1990. Chromosomal evolution in Malagasy lemurs: XII. Chromosomal banding study of Avahi laniger occidentalis (Syn: Lichanotus laniger occidentalis) and cytogenetic data in favour of its classification in a species apart – Avahi occidentalis. Am. J. Primatol. 21:307–316.

Rumpler, Y., Warter, S., Hauwy, M., Randrianasolo, V. and Dutrillaux B. 1991. Cytogenetic study of Hapalemur aureus. Am. J. Phys. Anthropol. 86:81–4.

Rumpler, Y., Warter, S., Hauwy, M., Meier, B., Peyrieras, A., Albignac, R., Petter, J. and Dutrillaux, B. 1995.Cytogenetic study of Allocebus trichotis, a Malagasy Prosimian. Am. J. Primatol. 36:239–244.

Rumpler, Y., Ravaoarimanana, B., Hauwy, M. and Warter, S. 2001. Cytogenetic arguments in favour of a taxonomic revision of Lepilemur septentrionalis.Folia Primatol. 72:308–315.

Rumpler, Y., Prosper, P., Hauwy, M., Rabarivola, C., Rakotoarisoa, G. and Dutrillaux, B. 2002. Chromosomal evolution of the Hapalemur griseus subspecies (Malagasy 64

Prosimian), including a new chromosomal polymorphic cytotype. Chromosome Res. 10:145–53.

Saint-Pie, J. 1970. Birth and rearing of a Brown lemur X Red-bellied lemur hybrid Lemur fulvus X L. rubriventer and breeding of Grey gentle lemur Hapalemur griseus at Asson Zoo. International Zoo Year Book.10:71.

Stanyon R., Koehler U., Consigliere S. 2002. Chromosome painting reveals that galagos have highly derived karyotypes. Am J Phys Anthropol 117:319-26.

Tagle, D. A., Goodman, M., and Miller, D. A. 1990.Characterization of chromosomes and localization of the rDNA locus in the aye-aye (Daubentonia madagascariensis).Cytogenet.Cell Genet. 54:43–46.

Tattersall, I. 1993. Speciation and morphological differentiation in the genus Lemur. Species, Species Concepts, and Primate Evolution, ed. Kimbel, W. and Martin, L., Plenum Press, NY, Pp. 163–176.

Todd, N. B. 1967. A theory of karyotypic fissioning, genetic potentiation and eutherian evolution. Mammalian Chromosome Newsletter 8:268–279.

Todd, N. B. 1970. Karyotypic fissioning and canid Phylogeny. J. Theor. Biol. 26:445–480.

Todd, N. B. 1975. Chromosomal mechanisms in the evolution of artiodactyls. Paleobiology 1:175–188.

Todd, N. B. 2000. Kinetochore reproduction underlies karyotypic fission theory: Possible legacy of symbiogenesis in mammalian chromosome evolution. Symbiosis 29:319-327.

Toder, R., O'Neill, R. J. and Graves, J. A. 1998.Chromosome painting in marsupials.I.L.A.R. J. 39:92–95.

Ventura, M., Archidiacono, N. and Rocchi, M. 2001.Centromere emergence in evolution. Genome Res. 11:595–599.

Warter, S., Randrianasolo, G., Dutrillaux, B. and Rumpler, Y. 1987.Cytogenetic study of a new subspecies of Hapalemur griseus.Folia Primatol. 48: 50–55.

Warter, S. Hauwy, M., Dutrillaux., and Rumpler, Y. 2005. Application of molecular cytogenetics for chromosomal evolution of the Lemuriformes (Prosimians). Cytogenet Genome Res 108:197- 203.

Wilson, E.B. 1925. The Cell in Development and Heredity. MacMillan NY.

Wurster-Hill, D.H. 1973.Chromosomes of the lemuridae. J. Hum. Evol.2:259.

Wyner, Y. M., Johnson, S. E., Stumpf, R. M. and Desalle, R. 2002. Genetic assessment of a white- collared x red-fronted lemur hybrid zone at Andringitra, Madagascar. J. Primatol. 57:51–66.

Yoder, A. D., Cartmill, M., Ruvolo, M., Smith, K. and Vilgalys, R. 1996. Ancient single origin for Malagasy primates. Proceedings of the National Academy of Sciences.93:5122–5126.

Yoder, A.D., Rasoloarison, R.M., Goodman, S.M., Irwin, J.A., Atsalis, S., Ravosa, M.J. and Ganzhorn, J.U. 2000. Remarkable species diversity in Malagasy mouse lemurs (primates, Microcebus). Proceedings of the National Academy of Sciences. 97:11325–11330.

Zaramody, A. and Pastorini, J. 2001. Indications for hybridisation between red-fronted lemurs (Eulemur fulvus rufus) and mongoose lemurs (E. mongoz) in northwest Madagascar. Lemur News 6:28–31.

CHAPTER 4

LEMURS AND SPLIT CHROMOSOMES

Kolnicki, R. L. 2011. Lemurs and split chromosomes in Margulis, L., Asikainen, C., and Krumbein W.E. eds. Chimeras and Consciouisness: Evolution of the Sensory Self. The MIT Press. Cambridge, MA. Pp. 173-181.

4.1. Introduction

“Despite repeated claims, there is very little evidence for gradual transition from one to another species. Here Kolnicki explains one of several proposed methods for rapid evolution: “karyotypic fissioning,” also called “centromere-kinetochore replication theory.” This theory focuses on the behavior of chromosomes and illuminates the history of a branch on our own lineage. Our 50- million-year-old primate relatives evolved into more than thirty species of monkey-like animals when, because of plate tectonics and spontaneous changes in their chromosomes, they became isolated on the great island Madagascar” (Margulis, Asikaiken, and Krumbein, 2011).

4.2. Lemurs of Madagascar

Madagascar, the fourth-largest island in the world, is located in the Indian Ocean off the southeast coast of Africa. It is home to a biota that is unique in all of life’s kingdoms. Many of its animals, chronicled in fables, are indeed fabulous. These include many species of lemurs that are found nowhere else in the world. They share characteristics not found among the other primates, such as female dominance and greater dependence on olfaction (sense of smell) over 67

sight. Unlike nearly all other primates, lemurs display cathemerality. It distinguishes them from nocturnal and diurnal mammals. Lemurs actively forage and socialize during portions of both day and night.

Lemurs, today classified in five families, were among the first suborders to diverge in the order Primata of our vertebrate class, Mammalia. This order includes lemurs, lorises, monkeys, orangutans, bonobos and other chimpanzees, humans, and other apes.

Lemurs may have colonized Madagascar as early as 80 million years ago or as late as 50 million years ago. Subfossil (recently extinct) lemurs found to have lived as late as 500 years ago were larger than the largest extant lemurs and the largest humans. Their recent extinction was probably influenced by hunting and habitat destruction. Today lemurs are endangered primarily because their natural habitats are becoming increasingly fragmented and diminished in size by slash-and-burn agriculture, livestock grazing, and logging.

Studies of the anatomy and the physical structure of fossil and living lemurs, along with behavioral and genetic analyses, have partly illuminated their evolutionary history. The fossil record alone does not help to determine the dates of divergence, because subfossil lemurs are part of the diverse contemporary fauna and not predecessors to modern lemurs. Some of the best evidence for the timing of divergence of lemur families, therefore, comes from genetic studies. Del Pero et al. (2006) used mitochondrial and nuclear DNA to investigate relationships among living lemur taxa, and 68

generated an acceptable phylogeny (figure 4.1). Although this family tree depicts reliable evolutionary relationship between the families, the processes that preceded diversification of lemurs from ancestral primates are still unknown.

Speciation, some involving divergence from one ancestral lineage into multiple descendant lineages, can result from various natural events, including the formation of geographical barriers. Geographical barriers (e.g., waterways, new mountains, volcanoes) lead to allopatric speciation, a gradual process whereby interbreeding is prevented when one population is physically separated into two or more populations.

One instance of allopatric speciation in lemurs occurred when the Betsiboka River formed a barrier to interbreeding and led to lineage branching in Eulemur, Lepilemur, and Propithecus species (Pastorini et al. 2003). Karyotypic fission is an alternative mode of speciation in mammals that occurs in sympatric populations (that is, within an interbreeding community) with no need for geographic isolation (Guisto and Margulis

1981).

4.3. Karyotypic Fission: Neocentromere Formation

Karyotypic fission is a cell process whereby each chromosome in a gamete (sperm or egg) fragments at the centromere/kinetochore region. The entire genetic content of the sperm or egg cell is inherited by the offspring cells, but it is packaged very differently than in the original parent. Large and few chromosomes give rise to smaller and more numerous ones. In my example (figure 4.2), one large chromosome generates four smaller ones. The macromolecular 69

sequences in the region of the chromosome are composed of DNA (centromere) and protein

(kinetochore) sequences, respectively. The spindle fibers (bundles of microtubules) attach to the centromere/kinetochores during cell division. The centromere/kinetochore is responsible for the proper segregation of chromosomes during mitotic cell division. Chromosomes move along the spindle fibers toward the poles of the cell. They are attached by their dynamic centromere/kinetochores to the microtubules.

Gametes contain only half of the full complement of chromosomes. Unlike body

(somatic) cells, gametes (egg and sperm cells) undergo two divisions: meiosis I and meiosis II. The first division separates chromosome pairs and reduces the chromosome number by half. The second, a mitotic division, separates sister chromatids. The result is four cells, each of which contains one copy of each chromosome. In ova, only one of these offspring cells is a functional gamete, an egg. The other three, significantly smaller than the egg, become non-functional polar bodies and disintegrate. Chromosome fission that occurs during the second division results in the separation of the chromosome halves. The molecular motors at the centromere/kinetochore region attach to the spindle molecules. Force is generated at these attachments. Chromosomes that possess the centromere/kinetochore at or close to their middles are called metacentrics.

Karyotypic fissioning that generates two smaller shorter, fissioned chromosomes with centromere/kinetochores near or at the end are called acrocentrics. If all metacentrics of a cell were to divide simultaneously, their offspring cells would inherit twice the number of chromosomes as in the parental cell. Matings shortly after the karyotypic

fission events between individuals with ancestral large metacentric chromosomes and individuals with a fissioned chromosome set are fertile.

An individual’s complete chromosome arrangement is easily visualized after representative cells are stained and photographed; the preparation is called a karyotype. Variations in karyotypes (variations in chromosome shapes and numbers) result from matings between individuals that have fissioned chromosomes and individuals that have retained the ancestral karyotype. Some offspring inherit whole chromosomes in combination with corresponding fissioned halves. A reduction in fertility is not likely before later heritable changes occur. The variety of inherited chromosomal arrangements is subject to mutation and further modification through natural selection. Certain crosses between unfissioned ancestors and newly fissioned descendants may be more successful than others. They may produce more offspring, which will carry specific new chromosomal arrangements. Other crosses may show reduced fertility, infertility, or other chromosomal incompatibility. This is one way speciation through reproductive isolation within a single population can occur.

Evolution through karyotypic fission has rejected or ignored because of the lack of an explanation of how fissioned chromosomes remain functional after “breaking.” My

“kinetochore reproduction” hypothesis describes the process by which fission results in viable but shorter chromosomes (Kolnicki 2000). Duplications of

kinetochore/centromeres survive. They are known to generate supernumerary kinetochore/centromere regions on a single chromosome. In fact, many chromosomes have “silent centromere sequences” where former functional centromere/kinetochores or partial centromere/kinetochores developed (Perry et al. 2004). Chromosomes with more centromere/kinetochores are preferentially distributed to functional gametes; those with fewer centromere/kinetochores fail to segregate into the egg and are distributed to the non-functional polar bodies of meiosis in females. In the case of chromosomes split between their duplicated centromere/kinetochore regions, both halves maintain full function for attachment of spindle microtubules. The sticky ends of

DNA are repaired by telomerase, an enzyme that is present in gametes.

Centric fission, or the transverse breakage of the centromere/kinetochore region, also may result in functional fissioned chromosomes (Perry et al. 2004). Cleavage at the centromere/kinetochore region leaves sufficient active sequences of centromeric protein and centromeric DNA attached to chromosome halves. The resulting chromosome fragments segregate normally. As with pre-duplication of kinetochore/centromeres, correct alignment with homologous regions on whole chromosomes during cell division is retained. Variations in karyotype can be maintained through backcrosses within the population.

4.4. Fission Driving Evolution

Neil Todd (2000) suggests that the variation of diploid numbers in mammals, such as in families of artiodactyls and in canids, resulted from karyotypic fission. The evolution of mammals does indeed follow a pattern, which is observable in the fossil record. Branching events (appearances of new taxa of animals) correlate well with changes in chromosomal complements. When major lineages diverged, chromosomal arrangements were modified in a manner predictable by application of karyotypic fission theory. A systematic increase in the number of chromosomes where a single long one was replaced by two shorter homologous ones occurred as mammalian lineages became more specialized. Todd hypothesizes that the original chromosomal arrangement consisted of mammals with fourteen large metacentric chromosomes. Among living mammals, the range in diploid number is 2n = 6–92. Most chromosomes in mammals other than the large X, the sex chromosome, exhibit signs of fission. Retention of the X relative to its fissioned descendants was selected for in reproduction. The long arm of the X chromosome is the same in most if not all placental mammals. Placental mammals tend to have the same large amount of DNA per chromosome. Whether or not the chromosome is one arm with telocentric centromere/kinetochores or two arms attached at the single metacentric centromere/kinetochore does not affect viability or fertility. The overall DNA content in marsupials’ X chromosomes is substantially less than that in eutherians. In placental mammals, the X chromosome is about 5 percent of the total genomic DNA.

Chromosome fission probably was important in the evolution of Marsupialia.

Both fission and single chromosomal fusions have contributed to the Y sex chromosomes of the kangaroo. In another marsupial, the swamp wallaby, females have

standard XX chromosomes, as do humans, but males carry a fissioned Y chromosome in addition to the typical X. (That gives males a karyotype of XY1Y2.) The monotremes

(platypus and echidna species, ancestors of the earliest mammals), which lay eggs as birds and reptiles do, show both the typical mammalian unfissioned karyotype (the karyotype of the duck-billed platypus is XX/XY) and presumably the subsequent fissioned pattern (the echidna females have X1X1X2X2, whereas the males have a fissioned X1X2Y). The general case remains that in most mammals the sex chromosomes are large and unfissioned whereas all other chromosomes (autosomes) have many different patterns of fission.

In the order Primata, Old World primates (including humans) have undergone at least three separate fission events. The first event, which occurred about 38 million years ago, may underlie the origin of different primate families of Old World monkeys and apes. This initial event generated diploid numbers of 2n = 40+, where the ancestor had diploid numbers of about 2n = 30+. As second fission event, about 20 million years ago, probably was the basis for the diversification of the hominoid group, which includes orangutans, gorillas, chimps, and humans. Diploid numbers increased from low numbers in early mammals to that typical of living primates (2n = 46–48) in the four ape groups. A separate, secondary fission event occurred in guenons, vervets, mangabeys, and some other Old World monkeys.

4.5. Fission in Lemurs

The diverse karyotypes found in the five living lemur families are explained by four separate fission events. Evidence for fission in evolutionary history can be gathered through application of different molecular methods to karyotypes. Chromosomes from animal groups that still retain the more ancestral condition (fewer and longer metacentric chromosomes) are matched to chromosomes of animal groups that have undergone fission. Using stains to distinguish regions

(bands) on chromosomes, the distribution of DNA banding patterns throughout the karyotype are compared. As in putting a puzzle together, chromosome patterns are reconstructed from representational clues to produce the most parsimonious ancestral arrangement.

The location of the centromere/kinetochore on chromosomes is also helpful in revealing clues about the history of fission and even the relative timing of the event. Through evolutionary time, the centromere/kinetochore on acrocentric chromosomes relocates again from the end of the chromosome toward the center (figure 4.1). This probably aids in chromosome stability during duplication and cell division cycles. From the inference that a karyotype in a more recent species of mammal has smaller, more numerous chromosomes with centromere/kinetochores at or near the center relative to a its ancestors we infer that a fission event occurred in the ancestors with the larger chromosomes long ago. Mammals whose body cells have karyotypes with smaller, acrocentric chromosomes are thought to have recently fissioned ancestors.

The five living families of lemurs are Daubentoniidae, Lepilemuridae, Lemuridae,

Cheirogaleidae, and Indriidae. Daubentoniidae comprises only one living representative species, the Aye-aye. Daubentoniidae was the first to diverge from a common ancestor and has a diploid number of 2n = 30. Lepilemuridae diverged second and comprises at least seven representative species. Lepilemurs have diploid numbers of 2n = 20–38. The

Cheirogaleidae, which diverged next, have diploid numbers of 2n = 46–66, followed by the Lemuridae, with diploid numbers of 2n = 44–62. The last to diverge were the

Indriidae, which display the highest diploid numbers among lemurs: 2n = 40–70.

Four fission events during the evolutionary history of lemurs explain both the diversity of chromosomal number and arrangement in living species. The ancestral condition probably was 2n = 20, which is still observed in modern lemurs (lepilemurs). A primary fission event before the diversification of modern lemurs generated a population of individuals carrying a variation of karyotypes. Daubentonia sp., which separated from the rest of the lemur families early, display chromosomes that are large and metacentric.

A separate primary event underlies the diversification between basal (earliest ancestors in this lineage) Lepilemurids and the rest of the lemur families. A secondary fission occurred some time before the divergence of the lemurids, the cheirogaleids, and the indriids. The basal population probably had a diploid number of 2n = 34, the minimum

number that could generate chromosomal numbers and arrangement observed in lemurids and cheirogaleids.

Finally, a subsequent fission event underlies diversification of the indriid group from a basal ancestor of around 2n = 40. Although fission can explain many of the modern karyotypes, multiple post-fission centromere/kinetochore relocations and mutations complicate the analyses. Indriid karyotypes are composed of numerous, short chromosomes. And many closely related species display their chromosomes at different stages of centromere/kinetochore relocation. This increases the difficulty of precisely reconstructing the timing and order of chromosomal change. However, a pattern can still be observed in the karyotypes across the family.

Karyotypic fission has formed the current chromosome patterns, speciation, and zoogeographic distribution in this lineage. Other, less important modes of evolution have participated, but chromosomal rearrangement via karyotypic fission is most consistent with molecular data. Lineages that diverged earlier maintain lower diploid numbers and longer chromosomes. They have not been subject to large-scale modifications. More recently evolved families of lemurs display unique patterns of increased chromosome number and, simultaneously, decreased chromosome size.

The theory of karyotypic fission explains evolutionary patterns in canids (dogs and cats), equids (horses), monkeys, apes, and artiodactyls (goats, sheep, deer). Chromosomal fission and relocation of centromere/kinetochores are significant to reconstructing the history of these mammalian taxa (Imai et al. 1986).

4.6. Might His Fissioned Chromosomes Be Sensed?

Reproductive isolation where genetic incompatibility leads to lack of offspring, or fewer offspring, or less fertile offspring puts selective pressure on populations to diverge. Mating between members of two diverging populations diminishes or ceases. Some mammals may be able to detect, presumably by olfactory clues, the fertility status of their potential mates.

Karyotypic fission affects some gene expression, particularly of genes located at the ends of chromosomes (telomeres). The closer a gene is to a telomere, the higher the probability of genetic mutations. Substitutions, deletions, or insertions of base pairs in

DNA alter the gene sequence and consequently change the protein structure. Genes that code for olfactory receptors, membrane proteins, or portions of proteins important for scent perception are located near telomeres in primates. Olfactory receptors in primates (including humans) show significant variation due to single-letter DNA mutation. More than 10,000 genes studied in chimpanzees were found to differ from their homologues in humans by approximately 8 percent. Much of this genetic variation involves genes and their protein products that effect reception of olfactory cues.

Observable behaviors indicate that odors (chemical olfactants) differently perceived by 78

these closely related primates have a genetic basis. We have seen that karyotypic fission alters chromosomes, and that the alterations include the generation of new centromere/kinetochores and telomeres. Although there is no evidence that karyotypic fission directly causes genetic and protein changes in primate olfaction, this idea deserves further investigation. Olfaction is significant to many aspects of primate social behavior, including mate selection, which suggests that chromosomal changes in olfactory receptors may contribute to divergence and/or stabilization of mammalian species.

The idea that the chromosome status of a potential mate’s fertility may be sensed invites research into direct correlation between chromosomal arrangements and mating behaviors. Fertility status indeed may be sensed, in general, in animal species, nearly all of which are unable to bypass two-gendered parent sexual reproduction by egg-sperm fertilization. Mammals, in particular, reproduce only through bi-parental sex. In some populations of subterranean naked mole rats (Spalex ehrenbergi), female rodents reject males of the same species that have differing chromosomal numbers (Nevo 1999).

Although female naked mole rats with high diploid numbers (2n = 60) tend to prefer males that have lower chromosome numbers, females with lower chromosome numbers (2n = 52, 54, or 58) prefer males that have the same diploid number as they do. The ability of mammals to assess the reproductive status (sterility, complementary gender, potential successful hybrids) requires detailed investigation. We can expect new

work on social behavior in the wild, on variations in karyotypes, on correlations with fossils, and on zoogeographic distribution of taxa to lead to more robust phylogenies.

We week a consistent reconstruction of lemur evolution in the Cenozoic era that includes all relevant information: molecular biological, biochemical, paleontological, geographic, behavioral, and (especially) chromosomal (karyotypic).

Figure 4.1: A phylogeny of the five lemur families with probable karyotype fission events depicted along evolutionary lines. Chromosome numbers of living descendants are in parenthesis.

Figure 4.2: Karyotypic fissions generate four functional descendants from a single ancestral chromosome through pre-duplication of the centromere/kinetochore components (genetic- DNA and protein-amino acid portions) of a chromosome.

4.7. References

DelPero, M., L. Pozzi, and J. C. Masters.2006. “A composite molecular phylogeny of living Lemuroid primates.” Folia Primatologica. 77:434-445.

Guisto, J. P. and L. Margulis.1981. “Karyotypic fission theory and the evolution of old world monkeys and apes.” BioSystems. 13:267-302.

Kolnicki, R. 2000. “Kinetochore reproduction in animal evolution: Cell biologicalexplanation of karyotypic fission theory.” Proceedings of the National Academyof Sciences. 97:9493-9497.

Godfrey, L. R. and J. C. Masters.2000. Comments on Kolnicki: Kinetochore reproduction theory may explain rapid chromosome evolution. Proceedings of theNational Academy of Sciences. 97:9821-9823.

Imae, H. T., T. Maruyama, T. Gojori, Y. Inoue, and R. Crozier.1986. Theoretical bases for karyotype evolution. The minimum interaction hypotheses.American Naturalist 128: 900-920.

Kolnicki, R. 2007. Chromosome change in the evolution of lemurs.In prep.

Nevo, 1999.Mosaic Evolution of Subterranean Mammals: Regression, Progression, and Global Convergence.Oxford University Press.

Pastorini, J., U. Thalmann, and R. D. Martin.2003. “A molecular approach tocomparative phylogeography of extant Malagasy lemurs.” Proceedings of theNational Academy of Sciences. 100:5879-5884.

Perry, J., H. Slater, and K. Choo.2004. “Centric fission – simple and complexmechanisms.” Chromosome Research. 12:627-640.

Todd, N. 2000. Mammalian evolution: Karyotypic fission theory. In Environemental Evolution: Effects of the Origin and Evolution of Life on Planet Earrth, ed. L. Margulis, C. Matthews, and A. Haselton, second edition. MIT Press. .

CHAPTER 5

CHROMOSOMAL CHANGES, ZOOGEOGRAPHICAL DISPERSAL

AND MAMMALIAN SPECIES ORIGIN

(LEMURS AND BATS)

Robin Kolnicki

University of Massachusetts

Department of Geosciences

Amherst MA

EVOLUTION FROM THE GALAPAGOS

Two centuries after Darwin

Professor Gabriel Trueba

University of San Francisco, Ecuador

editor

5.1. Abstract

The origin and geographical distribution of mammalian species of Madagascaran lemurscorrelate well with predictable chromosomal structural changes. The predictions, generated by karyotypic fission theory (KFT) include synchronous supernumerary centromere- kinetochores generated by duplication wherein two acrocentrics form from each single large metacentric chromosome in a karyotype. The tendency within any evolutionary lineage of mammals is for the diploid karyotype to proceed from ancient low numbers to more recent higher numbers. I explain here how the standard neo-Darwinian assumption that animal speciation is caused by natural selection of gradually accumulating random mutations in genes

(DNA sequences) provides a far less adequate concept of speciation than the "karyotypic fissioning theory" alternative, first recognized and detailed by Neil Todd. Hereditary changes in chromosomes, en masse centromere-kinetochore duplications, kinetochore-centromere relocation, and single pericentric inversions and fusions suggest new observations, predictions and most importantly, explanations of zoogeographic distributions amenable to testing by direct examination of the mammalian fossil record.

5.2. Introduction

A neo-Darwinian assumption that is widely taught in biology and evolution classes is that speciation is caused by natural selection of gradually accumulating random mutations in genes, i.e., changes in nucleotide base pair sequences in DNA that lead to the formation of new species in a branching tree-like pattern. The evolutionary process of mammalian speciation has not been documented in detail in the laboratory, the field or the fossil record. That speciation occurs so slowly is one reason given for the paucity of observation of gene changes corrrelated

with the origin of new animal species data. I claim that a more relevant fact is the general ignorance, with the notable exception of King (1995) in the many articles and books about speciation, relevant cytogenetic information tends to be systematically ignored. When a vast but poorly reviewed and co-ordinated literature on chromosomal change is properly evaluated zooogeographers and evolutionists will notice that the origin and geographical distribution of certain well-studied mammalian lineages (e.g., carnivores and artiodactyls, Todd; old world monkeys and apes (Giusto), lemurs (Kolnicki) correlate far better with chromosomal structural changes (KFT=karyotypic fission theory) to be discussed here than they do with the neo-

Darwinian assumption.

5.3. The theory and the lemur example

The application of karyotypic fission theory (KFT), developed primarily by Neil Todd (2000), to the mammalian infraorder Lemuriformes, the lemurs of Madagascar with their five families reveals the relationship of chromosomal features to mappable zoogeographical phenomena.

Structural chromosomal changes in mammal populations (related to the attachment of the chromosomes on the mitotic spindle) can be traced by cell studies. Karyotype analysis is a common practice that is done every day in laboratories such as those that take fetal cell samples to check for chromosomal anomalies (e.g., amniocentesis for Down's syndrome and other congenital abnormalities ="birth defects"). Karyotypes are photographs of all of the chromosomes found within the body cells of an animal. Photographs of chromosomes are cut out, aligned in order of size and numbered from largest to smallest. Karyotypes are published for significant representative species within mammal families and are therefore available for analysis. I show that the process by which lemurs evolved to their current geographical species

distribution and chromosome features is explained by a limited number of episodes of karyotypic fission (figure 4.2); a process where a range of chromosome numbers (up to double the ancestral number) evolves by means of an additional round ofkinetochore reproduction followed by chromosome fission (see Kolnicki, 2000). Karyotypic fission structurally changes the DNA packaging into smaller more numerous chromosomes without causing any significant change in DNA quantity or change in gene function.

My preliminary analysis, that mapped lemur chromosomes for only one family, Lepilemuridae

(figure 5.1) suggests that the origin of their diversification occurred at an ecological transition zone in Northern Madagascar between a humid evergreen forest along the East Coast of the island and the dry deciduous forest along the West Coast (Goodman, 2003). This geographical point of interest was once connected to both Africa and India before Madagascar moved southwestward to become an isolated continent.

The chromosome numbers found within related species when plotted on geographical maps will allow us determine the origin and evolution their chromosome diversity (figure 2). Banded chromosome studies have provided and are to be expected to provide much more useful information about potential fertility between individuals and they are significant for the identification of origin of reproductive isolation “speciation” within mammal families.

Geography plays an important role in generating the physical biodiversity found within a population. There is a vast amount of evidence that supports Darwinian theory that anatomical and physiological changes in animals result from selective pressure from the environment on gene expression. Genetic plasticity, such as changes in body size or coat color, common in

animals, reflects physical adaptation to specific environmental niche. Darwinian evolution, biodiversity, is often directly related to small changes in DNA; i.e. point mutations, duplications, and rarely deletions (or to gene regulation such as switching off genes by epigenetic methylation) that correlate to the great phenotypic or physical changes that we see in animals.

However, the reproductive isolation, “speciation”, of animals is here hypothesized to be attributable to gross changes in DNA structure (karyotypic fission followed by centromere relocation and sometimes fusion) and not to small changes in DNA sequence.

Chromosomally distinct groups (species) arise as fissioned chromosomes become fixed along a geographical cline. Chromosomes undergo predictable re-arrangement of the location of the centromere-kinetochore structure that governs movement of chromosomes during cell division.

The centromere/ kinetochore has a tendency to be located in the center or middle of the chromosome rather than be found on the chromosome ends. The repositioning of centromeres, centromere relocation (CR=kinetochore-centromere relocation), in small fissioned chromosomes reduces the fertility of the offspring that are chromosomal hybrids to a degree dependent upon the number of and combination of larger ancestral chromosomes and smaller fission-generated chromosomes that have undergone CRs (Kolnicki, 2000). Metacentric chromosomal clines are well documented in rodents (e.g. the house mouse, Mus musculus domesticus and the common shrew Sorex araneus) (Burt et al., 2009; Bidau et al., 2001). A single karyotypic fission event produces a range of chromosome numbers that spread within large continuous populations and it pre-adapts those populations for reproductive isolation due to centromere relocation. The fixation of the smaller metacentric chromosomes (that lead to reproductive isolation) occurs over a long time; probably during glacial refugia over several glaciations epochs (see Orlov, 2008).

In some species, chromosome fusions subsequent to fission have significantly contributed to isolation of populations. An example of speciation by multiple chromosome fusions is found in

Mus musculus domesticus chromosome “races” (2N=40 to 26) where established chromosome fusion in subpopulations lead to sterility between subpopulations, however, these same populations are fertile when crossed with the ancestral population that lacks fusion of specific chromosomes (see Capanna’s 1982 model for speciation and Baker and Bickham’s 1986 modification of that model in King, 1993). Mating behavior such as inbreeding within a subpopulation or formation of harem based social structure contribute to the fixation or stabilization of fissioned or fused chromosomes within a group. Fusion is apparently an exception rather a rule and I speculate that gender and age may play a role in the fusion of post- fissioned chromosomes where older individuals may have a decrease in DNA repair enzymes

(telomerase) found in gametes (sperm and eggs). Where there is sufficient evidence that telomerase enzyme repairs chromosome ends, I predict that the gametes (in particular sperm) in older individuals might have lower telomerase levels that result in stickier chromosome ends which lead to fusion.

5.4. The prospective bat example

Bats constitute some 20% of all mammal species. The two speciose mammalian taxa that I use here to illustrate the potential power of correlating Cenozoic mammalian zoogrography with chromosome evolution are: the mammalian orders Lemuridae (lemurs) and Chiroptera (bats).

Because all five lemur families are restricted to Madagascar, the continent island off eastern

Africa, and publications exist that apply chromosome theory to this naturally distinct,

geographically limited group, I only review and refer to literature in the case oflemurs.The zoogeographic distribution of lemurs is consistent with my kinetochore reproduction theory

(Kolnicki, 2000).

By contrast with the distinctive and limited tropical prosimian lemurs, there are about 900 extant species of bats and they are geographically dispersed on all continents of the Earth (with the exception of Antarctica). The feature that most distinguishes bats is that their forelimbs are developed as wings.Bats, because of their unique forelimbsare only mammals naturally capable of active flight (as compared to gliding).Given the fact that eighteen extant Chiropteran families are recognized I must restrict the discussion here to only a few groupsin well-studied taxa.

These are species in two suborders: Megachiroptera, "Old World fruit bats" or “Flying Foxes”, and Microchiroptera known as the "Ecolocating bats" or “True bats" (Koopman, 1993).

In the chiropteran order with its hundreds of species the diploid chromosome numbers range from a low of 16 (haploid N=9) to a high of 62 (N=31). Each family of bats predictably has chromosome complements that have a range limited to twice the lowest number of chromosomes (e.g. if a species has 20 chromosomes; then the greatest number of chromosomes in that family is predictably no greater than 40). There is a volume of printed data

(karyotypes) collected by numerous researchers readily available for further analysis (e.g. Atlas of mammalian chromosomes; (O’Brien, 2006). The chromosome numbers of all species within a single family provide useful evidence for reconstruction of that family’s evolutionary geographical dispersion. Geographical areas that have populations with a variety of high chromosome numbers (e.g. a range such as 32, 33, 34, 35, 36, 37, and 38 chromosomes found in a single reproductive population) are likely the location for the origin of species. Fixation of the post-fission smaller chromosomes is predicted to likely occur along geographical clines after 90

chromosomes spread from the fission epicenter. Populations that are closest to the location of the karyotypic fission event will have the greatest number of fissioned chromosomes whereas more distant populations are more likely to maintain all or most of the ancestral pre-fissioned chromosomes.

The fertility literature may be useful in assessing the validity of this idea. That a preliminary analysis of bat chromosomes suggests fission followed fusion (e.g. they have low chromosome numbers and a prevalence of chromosomes that have two centromere-kinetochores each) in one family necessitates further consideration of bat reproductive and social behavior. One of my purposes in this chapter is to enlist help. Only if I can interest other evolutionists and mammalogists in the powerful concepts of chromosome-change correlation with speciation, especially KFT, might the reasons for the enormous diversity and dispersed ranges of bats be constructed with confidence.

5.5. Geographical location of chromosome diversification

Based on KFT the geographical origin of a karyotypic fission episode should correlate with the highest chromosome numbers found within a taxon, in this case a family of bats. No geographical or physiological reproductive isolation phenomena are needed in the early stages following the karyotypic fissioning in the ancestral animals. This, the reproductive compatibility of ancestral unfissioned animals with fissioned members in the same interbreeding population has been observed (e.g., in pigs, carnivores, etc.). Note then, karyotypic fissioning has already been established as a phenomenon that underlies "sympatic speciation", a process that requires neither the geographic nor physiologic isolation of "allopatric" speciation.

Polymorphic chromosome numbers (e.g. a range such as 34, 35, 36, 37 and 38) found in a single population, in principle, indicates the location of a fission epicenter where chromosomal diversification arose. Chromosome numbers of descending order correlate to relative distance from fission epicenters as the fissioned chromosomes spread to neighboring allopatric populations and gradually, over time, became fixed due to inbreeding or other isolating factors.

Beyond lemurs and bats much chromosome data already shows that populations with lower chromosome numbers tend to be monotypic (i.e., all members of the population have the same low number of chromosomes).These monotypic populations are best considered to represent ancestral karyotypes, i.e., chromosome characteristics closer to the lower Cenozoic ancestral mammals rather than more recent species ("derived"). Therefore, I suggest that one can determine the geographical origin of species diversification by first identification of populations that are not monotypic for low or high chromosome numbers but those populations that show a range of high chromosome numbers.

Furthermore, I predict that the location of sympatric speciation events may be correlatable with geographical “zones of transition” (Wilkie, 1991). My approach, now underway, is to map the chromosome numbers of the Vesperitilionidae, Microchiropteran true ecolocating bats and determine the origin of their chromosomal diversification and subsequent geographical dispersion pattern. And then, with review the natural history; seasonal temperature and rainfall distribution and correlated vegetation patterns, geological and geographical history that chacterize the proposed karyotypic fission epicenters.One must ask if familial diversification of chromosome types occurs at geographical locations that experience significant times of transition with respect to extreme seasonal variation, changes in vegetation and other 92

ecological factors or even environmental evolution changes that have occurred over extended periods of time.It is encumbent on my colleagues and myself to investigate continental borders and tectonic plate movements and other major geological or climate changes that have affected and continue to affect the habitat of the mammals since their first appearance in the fossil record.

The next step in the analysis of extensive and complex speciation of chiropterans is to carefully enter on a world map the known karyotypic distribution details of bat chromosomes.Thechromosomal results may be superimposable on maps of geological and geographical history. They may reveal a discernable geographical distribution pattern that correlateswith enviromental transition zones (ecotones both actual and paleo-). I hope my analyses of lemur and bat chromosome evolution will be supplemented by analyses by others who study bat evolution. In fact since the task is so large (some 8000 to 10,000 estimated extant species of mammals, primarily rodents) one can look forward to analyses of other mammalian lineages.

In principle, karyotypic fission theory (KFT) explains patterns of speciation and global distribution (zoogeography) of mammals better than does neo-Darwinian theory. I develop the argument for the superior explanatory power of KTF theory relative to the vague and hard-to measure neo-Darwinian theory. The standard explanation is that one species changes by branching: from a single ancestor (the trunk of the phylogenetic tree) to two (or just one changed) descendant species. The new species appearance for neo-Darwinism involves gradual

accumulation of random mutations in genes (i.e., equivalent to random changes in DNA genes.

This neo-Darwinian assumption is equivalent to saying that new species appear by gradual accumulation of random mutations that are coded nucleotide base pair sequences in DNA molecules acted on by natural selection.)

The appearance of structural chromosomal change (karyotypic fission events followed by kinetochore-centromere relocation) even with the limited number of analyses now available explains the origins of new species and their zoogeographical dispersion patterns far better than the vague theoretical claim that animal species evolve by naturally-selected changes in ancestral

DNA sequences. Mapped chromosome numbers correlated to geographical dispersal patterns, in principle, need be tested by appropriate molecular biological studies. If mapped chromosome patterns are more consistent with other modern phylogenetic assessments, especially individual mutation-level DNA differences, then KFT must be seriously evaluated as a higher-level (larger visible hereditary difference than individual DNA-base pair mutational changes) mode for assessment of the origin, evolution and zoogeographic of mammalian species.

Whereas chromosomal changes that have been seen by thousands of competent microscopist- technicians are shown to isolate both populations and/or organisms within a single population reproductively from each other; mutations or changes in DNA sequences have not been clearly demonstrated to affect fertility in populations. Chromosome change can be directly measured at the geographical level in closely related mammal species or subspecies. (Wild boars preceded

domesticated pigs, wolves preceded dogs, chimpanzee ancestors presumably preceded Homo sapiens). In the study of relations of ancestors compared to descendants with known zoogeographical histories knowledge of DNA sequence details confirm close relationship of ancestors to descendants but do not illuminate either speciation or geographical distributions.

Karyotypic fission theory (KFT) on the other hand is supported by an immense poorly organized literature and permits reconstruction of mammalian past and current zoologeographic distributions.

Here is an example of the strength of the KFT theory (compared to the poorly defined and difficult-to-measure "random mutation" idea) in marsupials. The alternative that I challenge, that these marsupials evolved by accumulation of random mutations in DNA molecules, cannot be confirmed or disproved. It is best considered, in my opinion, as a very commonly held unjustified assumption. The "random mutation"-neo-Darwinian explanation just does not help explain the appearance of new mammalian species at a specific place and point in time. And how does the new species geographically distribute? Nor does neo-Darwinism explain how descendant species diverge? How do the new animals disseminate from common ancestors?

The "random mutation" neo-Darwinian claimed explanation: the "accumulation of random mutations in DNA molecules" does not generate experiments or observations whereas KFT details many of both. KFT not only explains what happened in the evolutionary history of modern animals but it helps predict what Cenozoic fossil and sub-fossil species should be found where and when in the geological record. DNA sequence analysis of kangaroos, koalas, wallabies, opossums or other didelphids show these animals to be more related to each other than to placental mammals but makes no clear statement about marsupial dispersion or 95

geographical history. But I emphasize that the conservative chromosome structure (e.g., diploid numbers of 2N=14) in early marsupials, for example, is useful in reconstruction of Cretaceous land masses by provision of additional evidence that the continents (Australia, South America) were still joined. Of course marsupials do not cross open ocean water.

Geographical distribution is important in the formation of biological diversity. A single species can inhabit a wide range and exhibit great diversity that is brought about by natural selection.

The Holarctic reindeer found in Scandinavia, Russia, China, Canada and Alaska (including caribou) are all a single species Rangifertarandus that exhibits variation in size and in coat pattern, changes brought about by adaptive selection by the environment or human selective breeding but they all have 70 similar chromosomes and they are all reproductively compatible.

There is a single species of reindeer yet geneticists subdivide Rangifer tarandus into at least eight “subspecies” which differ in certain genetic markers and mitochondrial DNA sequences

(Roed, 2005). Although, there is measurable DNA sequence divergence; there has been no

“speciation” as these circumpolar cervids are, in spite of their differences, like all humans today, genetically compatible. Furthermore, when we compare two large distinctive groups of mammals, marsupials and placental mammals we find similar specialized types convergently evolved morphological similarities (organs and organ systems) best suited to their environmental niche (e.g., digging, running, climbing, or flying). Analogous structures, features well suited to specific environmental niches, provide evidence that the landscape, geology, climate, and other geographical factors are important in the generation of biodiversity.

Geographical selective pressure contributes to biodiversity whereas gross changes in chromosome structure leads to reproductive isolation. Single base pair DNA-gene changes alone 96

do not correlate with names species or more inclusive taxa, geographic-environmental habitat features or other measurable biological patterns (Sapp, 2009).

Mammalian speciation is taught to occur by "the gradual accumulation of random mutations in genes (changes in DNA base-pair sequence) in populations that become geographically isolated" whereas KFT, that requires no geographic isolation, is mostly unknown, misunderstood or even dismissed without understanding. Based on a large international literature I can show that much evidence supports the idea that mammalian speciation events occur at specific geographical localities at identifiable geological times. These speciation events correlate with chromosome structural changes (in numbers of chromosomes and numbers of centromere- kinetochores on all of the chromosomes, i.e., KFT).

This chromosome-based theory (KFT) that was developed primarily by Neil Todd (2000) has not yet been applied to the huge mammalian order Chiroptera (bats). Distinguished by the modification of their forearms and hands as wings, bats represent about 20 per cent of all species of mammals. Bats comprise some 900 species distributed on all continents of the world with the exception of Antarctica (Nowak, 1994). The island continent of Madagascar alone harbors at least 18 species of these flying mammals (Nowak, 1994). I propose that the process by which bats evolved to their current geographical species distribution and chromosome features is better explained by karyotypic fission theory (KFT) than the generalization of accumulation of random mutations in a single lineage.

This kind of chromosome-zoogeographic study is entirely analogous to the phytogeographic- cytogenetic analyses routinely done by botanists to trace the evolutionary history of plants including adaptive radiation of potatoes, Solanum tuberosum family Solanaceae, nightshades from the Peruvian Andes; corn (Zea mays) family Graminae or Poaceae (grass family) from the

Mexican plateau or cabbage, cauliflower, mustard, cabbage, radish from the Greek seaside islands (Brassica, Rafinus, etc from the mustard family Cruciferae; or hexaploid (3 sets of diploid chromosomes) of cotton plants (Gossypium that dispersed from the Nile valley). Studies of polyploidy based on cytological analyses in plant evolution have been far more important than

DNA sequence analysis for phytogeography. Why are not polyploid analyses made for zoogeography the way they are for plants? Because variation in ploidy (where entire SETS of chromosomes are duplicated or triplicated) is almost always lethal in animals. Whereas polyploid mammals most often will not survive to reproduce, polyploidy is common in plants and especially important in the interpretation of speciation and phytogeography.

Whereas gradual accumulation of random mutations in DNA -claims for a speciation mechanism is vague and unsubstantiated (both in plants and animals) KFT (analogous to polyploidy phytogeographic analyses in botany) provides precise and testable predictions. These predictions can be verified by studies I recommend of chromosome numbers and structure

(including centromere-kinetochore reproduction) in mammals, especially those with defined, limited habitats and geographical ranges (e.g., Madagascar lemurs and certain families of bats;

Vespertilionidae, “evening bats” and Phyllostomidae, “new world leaf nose bats” and old world

Pteropodidae “fruit bats”). Once related to behaviors, habitats and the geographical distribution of the animals throughout their ranges, valuable clues to paleoenvironments can be collected. The size, morphology and number of chromosomes found within bats and other mammalian taxa follow predictable patterns with respect to current zoogeographical distribution.

The neo-Darwinist claim, on the other hand is too imprecise and ill-defined to put to a comparable tests. A literature review of the contribution of DNA mutations to speciation in mammals predominantly yields speculation about the significance of changes in protein production that effect phenotype (physiological changes sometimes alter the physical appearance of an organism such as a differing coloration) but does not offer a significant plausible mechanism to explain speciation within a population. In other words, genetic changes often lead to increased diversity but they do not contribute significantly to speciation in animals.

Figure 5.1: Biogeographical distribution of Lepilemur chromosome numbers. The ancestral arrangement of twenty chromosomes in Lepilemur ruficaudatus is found is on the west coast of Madagascar. This origin of lemurs on the western coast is consistent with the widely held view that ancestral prosimian primates rafted over from the east Coast of Africa. A “fission epicenter” is found in the north of the island at a “transition zone” located between a dry evergreen forest and a wet rainforest. Fissioned chromosomes spread through interbreeding populations along the periphery of the island and became “fixed” when centromere- kinetochore relocation caused reproduction isolation, “speciation”.

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5.6 References

Bonaccorso, F.J. 1998. Conservation International Tropical Field Guide Series: Bats of Papua New Guinea. Conservation International, Washington, D.C.

Burt G, Hauffe HC, Searle JB. 2009. New metacentric population of the house mouse (Mus musculus domesticus) found in Valchiavenna, Northern Italy. Cytogenet Genome Res. 125(4):260-5.

Bidau CJ, Giménez MD, Palmer CL, Searle JB. 2001. The effects of Robertsonian fusions on chiasma frequency and distribution in the house mouse (Mus musculusdomesticus) from a hybrid zone in northern Scotland. Heredity. 87(3):305-13.

Dolan, M.F., Melinitsky, H., Margulis, L., Kolnicki, R. 2002. Motility Proteins and the origin of the nucleus.The Anatomical Record 268:290-301.

Dolan, M.F. 2005. The missing piece: the microtubule cytoskeleton and the origin of

eukaryotes, in Microbial Phylogeny and Evolution: Concepts & Controversies. Sapp, J., ed.

Giusto, J. P. and Margulis, L. 1981. Karyotypic fission theory and the evolution of old world monkeys and apes.BioSystems 13:267-302.

Joblonka E., and Lamb, M.J. 2006. Evolution in Four Dimensions: Genetic, Epigenetic, Behavioral, and Symbolic Variation in the History of Life. The MIT Press, Cambridge, MA.

King, Max. 1995. Species evolution: The role of chromosome change. Cambridge Univ Press, Melbourne Australia.

Kolnicki, R.L. 1999. Karyotypic fission theory applied - Kinetochore reproduction

and lemur evolution. Symbiosis 26: 123-141.

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(Invited commentary by Godfrey, L.R., and Masters J.C. 2000 Kinetochore reproduction theory may explain rapid chromosome evolution. Proceedings of the National Academy of Sciences of the United States of America 97: 9821-9823.)

Kolnicki, R.L. 2009. Kinetochore reproduction (=karyotypic fission) theory:Divergence of lemur taxa subsequent to simple chromosomal mutation.

In review for Evolution)

Kolnicki, R.L. 2010. Lemurs and split chromosomes inMargulis, L. Asikainen C. and Krumbein, W.E., eds. Chimeras and Consciousness: Evolution of the Sensory Self (in press). Cambridge: MIT Press

Kolnicki, R. L. 2010 .Vespertilionidae (Chiroptera) chromosomal evolution explained by karyotypic fission theory (manuscript, work in progress).

Koopman, K.F. 1993. Chiroptera in Wilson, D.E., and D.M. Reeder, eds. Mammalian species of the World, 2nd ed., Smiths. Inst. Press, Washington, D.C., pp. 137-241.

Kuhn, T. 1996. The structure of scientific revolutions (3rded.). Chicago, IL: University of Chicago Press

Margulis, L. and Sagan, D. (2002) Acquiring genomes: A theory of the origins of species. New York, NY: Perseus Books Group (Basic Books)

Margulis, L. and Chapman, M.J. (2010) Kingdoms & Domains

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Neuweiler, G. 2000. The Biology of Bats. New York, NY: Oxford University Press.

O’Brien, S.J., Menninger, J.C., Nash, W.G. 2006. Atlas of Mammalian Chromosomes.John Wiley & Sons, Inc. Hoboken, NJ.

Orlov VN, Kozlovskiĭ AI, Balakirev AE, Borisov IuM. 2008. Fixation of metacentric chromosomes in populations of the common shrew Sorex araneus L. from populations of Eastern Europe. Genetika. 44(5):581-93.

Roed, Knut H. 2005.Regugial origin and postglacial colonization of holartic reindeer and caribou. Rangifer, 25 (1): 19-30

Russell A.L., Ranivo J., Palkovacs E.P., Goodman S.M., Yoder A.D. 2007. Working at the interface of phylogenetics and population genetics: a biogeographical analysis of Triaenops spp. (Chiroptera: Hipposideridae). Molecular Ecology Feb;16(4):839-51

Todd, N. 2000. Karyotypic fission theory in Margulis L., Matthews C. & Haselton A., eds. Environmental evolution: Effects of the origin and evolution of life on Planet Earth, second edition. Cambridge: MIT Press.

Nowak, R.M. 1994. Walker’s Bats of the World. Baltimore, MD: The John Hopkins University Press.

Sapp, J. 2009. The New Foundations of Evolution: On the Tree of Life, Oxford University Press.

Villasante A, Méndez-Lago M, Abad JP, Montejo de Garcíni E. 2007. The birth of the centromere.Cell Cycle. Dec; 6(23):2872-6.

Wilkie, R.W. 1991. “New England: A Region of Convergence and Ecological Diversity” in Historical Atlas of Massachutestts.Ed.s R. Wilkie nd J. Tager. The University of Massachusetts Press.Amerst.Overview chapter, pp 2-3.

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White, M.J.D. 1973. Animal species and evolution. Cambridge University Press,

Cambridge, UK.

Zubaid, A., McCracken, G.F., Kunz, T.H. 2006. Functional and Evolutionary Ecology of Bats. New York, NY: Oxford University Press.

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CHAPTER 6

RANGIFER TARANDUS RUMEN MICROBIAL SYMBIONTS PERMIT HOMO SAPIENS SURVIVAL IN

EXTREME WINTER ARCTIC ENVIRONMENT

6.1. Abstract

Reindeer, cervids adapted to live on a limited fibrous diet in extreme arctic biomes have coexisted with nomadic humans. These ruminants depend upon a specialized symbiotic microbial community within their digestive system. I infer that during extreme arctic winter conditions Pleistocene humans as do contemporary people obtained nutrients in the Paleoarctic indirectly via Rangifer from a diet limited to plant cellulose and lichens. The seasonal changes in the rumen symbionts are described. The microbial community, which consists of primarily anaerobic bacteria and ciliate protists facilitates the digestion of lichens indigestible to inhabitants of more southern temperate environments. Circumpolar people co-evolved with

Rangifer and their symbionts for tens of thousands of years across the Eurasia and North

American arctic.

6.2. Reindeer evolution

All extant reindeer (Rangifer tarandus) are taxonomically assigned to the Mammalian suborder

Ruminantia as Cervidae (deer family), a suborder that first appeared in the Eocene fossil record,

50 million years ago (Hackmann and Spain, 2010). The word “ruminant” is derived from Latin ruminare, i.e. to muse or chew over again. All ruminants, including cattle, bison, sheep, goats, antelopes, and giraffes repeatedly regurgitate and re-chew food. They have pregastric 105

fermentation chambers (i.e. rumen, reticulum and omasum) specialized for the digestion of roughage. Ruminants are unique in their production of two enzymes, stomach lysozyme and pancreatic ribonuclease which aid in digestion of bacteria and degradation of bacterial RNA

(Barnard, 1969; Dobson et al., 1984). That reindeer are classified with deer is consistent with large scale intergeneric analysis which applied genome-wide single nucleotide polymorphisms

(SNPs) (Decker et al., 2009). Analysis of mitochondrial DNA suggests reindeer appeared 13.6 to

15.4 million years ago (Randi et al., 1998).

All ruminants are grouped according to their foraging method (Hofmann, 1984). Browsers that select tender leaves and herbage first appeared in the middle Paleocene and expanded during the late Eocene (Collinson and Hooker, 1991). Modern browsers including deer, moose, and giraffe have less developed relatively smaller rumens than those of roughage eaters; they are less tolerant of a high fiber diet (Hofmann, 1984). The small rumen size and weaker reticulorumen muscles in browsers may enforce their avoidance of grass forage (Clauss and

Hofmann, 2008). Grazers such as cattle, buffalo, sheep, camels, and antelopes by contrast have stronger chewing muscles and rely heavily on rumination. Grazers are better adapted to eating fibrous hay; they have less trouble with gastrointestinal-tract disorders in captive zoo environments (Clauss et al., 2003a). Grazers appear in the Miocene during the expansion of grasslands (Thomasson and Voorhies, 1990). A shift from browsing on trees to grazing on grasses that grow near water holes may have been evolutionarily selected for during extensive dry climate periods when animal populations become geographically isolated by their dependence upon water proximity (Derry, 2009). Grazers in general, are more dependent on open water (Western, 1995). A broad range of modern, water-dependent species in Africa are 106

typically reported to be at distances less than 10km from water holes during dry seasons (Derry,

2009). Reindeer share the characteristics of both grazers and browsers. They are highly adaptable to varying environments and changing habitats and are classified as intermediate feeders along with elk, pronghorns, impala, gazelles and eland (Cheeke and Dierenfeld, 2010).

Nowegian reindeer and Svaldbard reindeer have similar digestive systems, however, the

Svaldbard reindeer has a larger distal fermentation chamber. Dietary differences are associated with variations in rumen anatomy and physiology.

All of the reindeer in the world (including Alaskan caribou) comprise a single species (Rangifer tarandus) but several subspecies are recognized (Hummell & Ray, 2008). Reindeer evolved in glacial refugia during the late Pleistocene (Sommer and Nadachowski, 2006). Variation between geographically distinct populations of reindeer does occur; body size is smaller or differences are seen in antler morphology or fur coat pattern. These phenotypic (physical) differences among living reindeer are attributed to adaptive responses to post-glacial environmental change

(Flagstad and Røed, 2003). An exception of the North American woodland caribou, which based on mitochondrial DNA (mtDNA) analysis, likely evolved in refugia (Røed, 2005). Caribou survived and adapted to harsh arctic conditions through four cycles of continental glaciations over the last two million years (Hummel and Ray, 2008). Habitat, behavior and diet distinguish three caribou ecotypes, namely migratory tundra, boreal forest, and mountain caribou (Hummel and

Ray, 2008). Mountain caribou living in deep snow forage more heavily on arboreal lichens rather than on terrestrial lichen mats buried beneath snow. All wild caribou in North America and both wild and domestic reindeer in Eurasia are one single species comprised of at least eight

“subspecies” that differ in certain genetic markers and mtDNA sequences (Røed, 2005). In all 107

reindeer the diploid chromosome number is 70 (Gripenberg et al., 1986). Although, there is measurable DNA sequence divergence; physical diversity is limited and no speciation or reproductive genetic incompatibility has been reported in this large group of mammals.

6.3. Reindeer–human association

Holarctic reindeer (Rangifer tarandus) and humans have a long history of close association in extreme cold northern locations. Reindeer are found from 46° to 80° North latitude in Norway including Svaldbard, Sweden, Finland, the Kola Peninsula, Russia, China, Canada and Alaska

(including Alaskan caribou). Reindeer played a significant role in human colonization in the arctic and sub-arctic at the end of the last glacial period. Ancestral reindeer ranged across vast areas of tundra in Eurasia and North America during their original demographic expansion

115,000 years BP (Røed, 2005). For tens of thousands of years, reindeer have been a major resource for human populations in northern North American and Eurasia (Burch, 1972). Antlers and bones of reindeer dating 12,500 14C year BP are the most frequently found vertebrate remains from the late glacial deposits of Southern Scandinavia (Aaris-Sorensen et al., 2006). In modern relationships, Sami nomadic herders fight legal battles over land usage rights while they continue to tend reindeer that provide them supplementary meat (Einar, 1997). Sami herders accompany reindeer and guide them to sustainable grazing during lengthy migrations to their inland winter lichen feeding grounds and to summer coastal grazing areas (Kalstad, 1997).

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The reindeer-human association probably permitted the earliest colonization by any primate into permafrost tundra in post Pleistocene glacial period. Reindeer enabled humans to survive extreme cold environments during times of severely depleted food sources and provided insulated clothing essential for their survival. Scandinavian Sami, Russian Nenets, and Chinese

Evenki reindeer herders maintain historically rich cultures long connected to the reindeer.

Norwegian Sami recognize eight annual seasons defined by cyclical variation in weather to which reindeer herds respond: migration, foraging, reproductive behavior (Kalstad, 1997). Sami follow the herd during spring parturition season as the females return to same localities each year to calve. Winter foraging depends on snow type and ice coverage. Limitations in quantity and quality of plant food force reindeer to migrate. Reindeer have evolved an extraordinary rare ability to see into deep ultraviolet wavelengths, a behavior that better enables them to locate lichens in the snow (Hogg et al., 2011). They also avoid predators by visually detecting urine in the snow. Nomadic Sami have an extensive language which describes the snow and ice conditions essential to reindeer herding to suitable foraging grounds. They avoid ice and deep snow covered pastures (Jernsletten, 1997). Depending on season and on geographical location, modern Sami lead reindeer either to inland mountain forests away from cold frozen coasts or towards coastal pastures of grasses.

Lichen and reindeer population dynamics are evolutionarily interdependent. Reindeer herds potentially enlarge as a function of lichen abundance. However, over-foraging is detrimental to lichen mat bio-density and it can extirpate reindeer populations (Klein and Shulski, 2009). The relatively smaller Svaldbard reindeer have lost dietary lichens due to overgrazing and trampling and now no longer include them in their winter diet (Mathiesen, et al., 2005). Sustainable 109

grazing guided by Sami reindeer herders promote the growth of fruticose lichen mats (Gaare,

1995). Lichens establish colonies on near bare rock. Lichen overgrowth and decay leads to the formation of humus that has sufficient water storage capacity to support the succession of ferns, shrubs and other vascular plants (Cornelissen, 2004). Sustainable grazing ensures lichen growth is not inhibited by the shade of vascular plants and therefore a lichen sward is available for future nourishment.

6.4. Reindeer diet

Reindeer have a diverse, unique arctic diet. Reindeer forage in general on a variety of lichens, mushrooms, grasses and sedges, horsetail, herbs, twigs, bushes or trees. Free-ranging

Scandinavian reindeer select their nutritional sources from a variety of approximately 250 species plus 200 additional occasional plants (as described by Skuncke, 1958 in Westerling,

1970). They forage on lichens that grow on trees, stones and on the ground (Inga, 2007). The three preferred lichen mats are Cladonia stellaris (up to 15 cm thick); Cladonia rangiferina,

Cladonia mitis (pastures 3-7cm thick), and Sterocaulon paschal in heavily grazed areas

(Westerling, 1970). Sami herders, when interviewed, claim Cladonia genus as the first choice of the reindeer (Inga, 2007). Trees and bushes are foraged year round. In winter Scandinavian reindeer feed on willows (Salix spp.), mountain birch (Betula tortuosa), mountain ash (Sorbus aucuparia) and aspen (Populus tremula) (Westerling, 1970). During warmer seasons reindeer are able to feed on a diverse variety of available plant species including blueberries (Vaccinium myrtillus) and grasses (e.g. wiregrass, Deschampsia flexuosa that grows along streams).

Individuals within the same population may have distinctive rumen microbial communities that reflect their personal food preferences during seasons of abundance (Westerling, 1970).

Svalbard reindeer do not have access to lichen; their diet is dominated by the purple saxifrage 110

(Saxifraga oppositifolia) a common arctic angiosperm; grasses, sedges, shrubs, herbs and mosses are also eaten (Sørmo et al, 1999).

6.5. Reindeer rumen research

Community composition of reindeer rumen microorganisms includes research by several authors (Westerling, 1970; Opin et al., 1985; Aagnes and Mathiesen 1995; Imai et al, 2004;

Sundset et al., 2008, 2009). The first studies of reindeer rumen microbial community were done in 1895 in the Zoological Gardens of Berlin by R. Eberlein who hypothesized that the other ruminants in the zoo had influenced the rumen microbiota of the captive reindeer (Westerling,

1970). Free-living Scandinavian reindeer slaughtered for food provide an abundant source of rumens for analysis and for comparison of their microbial communities. Rumen fluid is obtained in living animals by means of temporary insertion of an esophageal tube or by surgical insertion of a fistula. Fistulated reindeer in Alaska provide extensive data about rumen digestion in captive reindeer (Holleman et al., 1979). A surgically implanted window provides easy access to samples of ingested food and the rumen microbes.

6.6. The Rangifer rumen stratified microbial community

Within the rumen, ingested materials stratify into layers. Rumen stratification is more pronounced in large grazing species (e.g. musk oxen) than in browsers; buoyant grass floats on the dorsal surface and fine particles settle to the ventral surface (Clauss et al., 2003b, 2009). In semi-domestic Finnish reindeer, indigestible sand and small rocks settle below a thick layer of finely chewed lichen and herbage in the rumen (figure 6.1) (described by Holflund and

Nordqvist, 1961 in Westerling, 1970). The chewed natural feed of lichen and herbage is rapidly soaked and stratifies at the bottom of the rumen (Westerling, 1970). Above is a liquid enriched

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with microscopic life adhering to tiny fermenting food particles. This fine liquid flows into the reticulum and is digested. On occasion reindeer ingest grass or hay; the straw will float dorsally upon the liquid layer. Gases rise to surface and are eructed (i.e. “burped up”). Large quantity of bacteria and ciliates digest the cell walls of plants and extract energy from cellulose. When microbes spill from the rumen (a symbioorgan, a hypertrophial esophagus) into the true stomach their bodies are digested. The resulting amino acids and small peptides that are absorbed by the reindeer are hence derived from these microbes (Hobson and Stewart, 1997).

6.7. Reindeer symbiotic microorganisms

Reindeer depend upon a complexcommunity of microorganisms to digest their food. The complete breakdown of carbohydrates to smaller molecules, methane and carbon dioxide, is a multistep process that involves many organisms. Ingested plants, mushrooms and lichens travel from mouth down the esophagus to the rumen, a blind anoxic habitat enriched with a microbial community dominated by anaerobes such the large protist Epidinium gigas which has been observed ingesting the lichen particles (Westerling, 1970). The richly diverse microbial rumen community that provides essential nutrients, protein, and short chained fatty acids to the reindeer are listed in table 6.1. Holotrichs, protists that have a relatively uniform pattern of cilia coving their bodies, are able to utilize soluble carbohydrates while entodiniomorphs, recognizable by the ciliary band around their mouth, engulf starch granules and bacteria

(Cheeke and Diernefeld, 2010). Bacteria and protists, unlike mammals, synthesize digestive enzymes to break down the cellulose of vascular plant cell walls as well as the complex polysaccharides of lichens. Distinct microbial communities within the rumen (foregut), small intestine (midgut), and lastly cecum colon complex (hindgut) respond to nutrient availability.

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Specialized prokaryotes degrade cellulose (e.g. Butyrivibrio fibrisolvans), starch (e.g.

Ruminobacter amylophylis), lactate (e.g. Selenomonas ruminatium subsp. lactilytica in the small intestine of young reindeer), pectin (e.g., Lactinospira multarus), xylan, protein and urea (Orpin, et al., 1985). Since fermentating microbes break glucose to pyruvic acid and volatile fatty acids the reindeer intestine absorbs very little glucose. The short-chain fatty acids, absorbed by the rumen papillae, provide in general for ruminants the main energy source (Cheeke and

Dierenfeld, 2010). The resulting gases, primarily methane and carbon dioxide are eructed by reindeer. Within the rumen, anaerobic fungus-like chytridiomycotes invade fibrous plants and begin hydolysis of polysaccharides including partial degradation of lignin, hemicellulose, and pectins (Gordon and Phillips, 1998). Chytridiomyotes are frequently referred to as fungi however, their zoospore utrastructure diagnostically places them in the Phylum

Chytridiomycota of the Protoctista Kingdom (Margulis and Chapman, 2010). Zoospores breakdown lignified cell walls making plant degradation more accessible for cellulytic bacteria

(Cheeke and Dierenfeld, 2010). Microbes that provide the reindeer with such nutrients as fatty acids (acetic, propionic, and butyric acid) absorbed through the rumen wall, lining of the stomach and the blood may provide as much as 70% of the daily energy requirements for reindeer (Hungate, 1966; Annison and Armstrong, 1970).

Reindeer utilize protein products of anabolism and digestion from their fermentative microbes.

Some bacteria use ammonia as a nitrogen source to synthesize amino acids. Ruminants ingest copious amounts of saliva which provide urea, sodium, potassium, phosphate and bicarbonate ions to the rumen (Cheeke and Dierenfeld, 2010). Ureolytic bacteria adhere to the rumen wall and provide ammonia for protein synthesis to bacteria in the rumen fluid (Mathiesen et al., 113

2005). Rumen protists (ciliates) depend upon bacteria for supply of nitrogen (Margolin, 1930).

Predation by ciliates accounts for 90% of the eubacterial protein turnover in the rumen

(Newbold et al., 1996). Protein is obtained by protein synthesis by ciliates and by the passage of microbes into the acidic abomasum (true stomach) that has a pH of 2.7 - 3.5 where they are killed and then digested in the small intestine.

Free living reindeer all have in common a similar rumen microbial community. Finnish reindeer all have 19 regular species of ciliates that vary in numbers according to season (Westerling,

1970). Reindeer in China, Russia, Finland and Alaska all share a similar ubiquitous rumen microbial community of 18 species of protists in their rumen fluid samples suggesting that

Rangifer tarandus has been isolated from other ruminants for a long time (Imai, 2004). An exception is that Svaldbard reindeer have only entodiniomorphid ciliates. They lack the holotrich ciliates found in other reindeer; perhaps due to starvation resulting from poor winter forage (Williams and Coleman, 1988). Reindeer rumen microbes co-evolved with a specialized arctic diet of lichens, mushrooms, mosses and other foods that lie buried beneath snow throughout much of the year. The number of each species of protist, bacteria, or fungi within any individual ruminant corresponds to the specific diet of the animal at a specific time

(Westerling, 1970).

6.8. Seasonal changes

Seasonal changes in weather are inevitably accompanied by vegetation changes and the microbial community inside the rumen responds. In mountain Sami villages in Northern

Sweden, snow covers the ground from October 1st through the second week of May (Inga,

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2007). Changes in the percentage of grasses, woody plants, lichens and mosses ingested by reindeer as they migrate between summer and winter pastures influence gastrointestinal microbiota and metabolism (Mathiesen, et al., 1999). Colder arctic seasons lead to diminished green vegetation and there is a corresponding change in populations of rumen microorganisms, a shift in dominant species that are best able to digest the predominant food type from tender spring vegetation to toxic winter lichens. Ciliate numbers are highest in late winter when reindeer forage heavily on lichens (Sundset et al., 2009). However, ciliates decrease in winter when food is limited (Westerling, 1970). Mean population densities of rumen methanogenic archaea, eubacteria and ciliates in Norwegian reindeer during summer were 3.17x109, 5.17x1011 and 4.02 x 107 (numbers per gram wet weight), respectively (Sundset et al., 2009). Starvation for 3-4 days results in as much as a 99.7% reduction in cultivable anaerobic rumen bacteria, and alters the composition of the microbial community and its ability to digest cellulose (Mathiesen et al., 1984; Aagnes et al., 1995; Olsen, 2000). Rumen ciliates decrease 75% after 3 days starvation (Mathiesen et al., 1984). Starvation, of course, has a detrimental effect on microbial population counts and it can complicate seasonal rumen comparisons.

In a habitat unlivable for most large mammals throughout the annual seasonal changes arctic ruminants such as the reindeer undergo seasonal changes in their highly specialized digestive system well adapted to an extreme arctic diet. Seasonal variation in ciliate (protist) and bacteria populations within the reindeer rumen and cecum microbial communities are shown in Table

6.2. During winter, the food source for many reindeer populations may be limited to mostly lichens that contain secondary compounds that are potentially toxic (e.g. fumaric acid) to both humans and reindeer. The rumen microbes digest lichens and detoxify the lichen metabolites 115

(Sundset et al., 2010). The reindeer’s cecum, located at the distal end of the large intestine prior to the colon, harbors seasonally changing methanogic bacteria (Mathiesen et al., 1987).

Although, microbial cells found beyond the gastric stomach cannot digested, their microbial products are absorbed and provide an important source of nutrients to the reindeer (Mackie et al., 2000).

When reindeer feed on tender green sprouts in the spring and leafy plants and seeds in the summer; their rumen bacterial populations increase. Cellulolytic and starch utilizing species increase relative to the availability of plant based nutrients. High-arctic Svalbard reindeer rumen wall tissue viewed under scanning electron microscope (SEM) show more bacteria cover the rumen wall in the summer than in the winter. Rumens collected during summer months have nearly 30% of the epithelium covered by bacteria; significantly Ruminococcus sp. coated with sticky glycocalyx which help them adhere to plant cell walls and to rumen epithelium

(Cheng and McAllister, 1992). In winter only 10% of the rumen epithelium is covered with smaller bacteria that do not have a glycocalx. Butyrivibrio fibrisolvens and Streptococcus bovis dominate the summer Svalbard reindeer rumen community and their numbers are four times greater than during winter (Orpin, et al., 1985). B. fibrisolvens isolated from Svaldbard and

Norwegian reindeer have been shown to solubilize cellulose (Aagnes et al., 1995; Olsen and

Matheisen, 1998). Reindeer harbor high concentrations of Bacteroides, Fibrobacter,

Streptococcus and Clostridium (Orpin et al, 1985). Free range and captive fed reindeer vary in microbial populations. Captive reindeer fed lichens ad libitum have greater numbers of

Streptococcus and Clostridium species. Bacterial populations respond to changes in the reindeer’s diet. 116

The quantity and relative ratios of ciliate species are influenced by the availability and type of nutrients ingested. The rumen protist, Entodinium caudatum produces chitinolytic enzymes for mushroom digestion (Moravi, et al., 1996). Alaskan reindeer forage on mushrooms for protein in the autumn (Boertje, 1990). Many Entodinium spp. diminish in winter; they likely specialize on starch to supply energy. However, one of the most characteristic ciliates of the reindeer rumen, Entodinium anteronucleatum is abundant during lichen season; it possibly depends on energy from lichens (Westerling, 1970). Entodinia (except small species) increase during winter and are lowest in summer. Larger species, Entodinium longinucleatum and E. dilobum have been found containing smaller entodinia, which they apparently digest (Westerling, 1970).

Entodinium bursa ingests the smaller Entodinum caudatum and bacteria as a source of amino acids for protein synthesis (Coleman and Hall, 1984). Entodiniaprovide peptides and amino acids to reindeer as they are in turn digested. Population densities of diplodinia ciliates increase in autumn as reindeer gain weight in preparation for winter; and their diets show increases in plant fiber and cellulose.

In winter when protein-rich food is severely restricted, reindeer critically depend on rumen symbionts for a protein source. The most common diagnosis in free-living reindeer found dead in winter without trauma is inanition when fat deposits are completely depleted (Josefsen et al.,

2007). Svaldbard reindeer use fat reserves to provide only 10-30% daily energy requirements, but the remainder must be supplied through digestion via ruminal microorganisms (Orpin, et al.,

1985). During winter the reindeer secretes considerable amounts of urea into the rumen

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contents that supplies additional nitrogen to rumen microbes (Orpin, et al., 1985). The reindeer’s winter food, approximately 85% lichen is digested by protists. Lichens provide highly soluble carbohydrates, low crude fiber and cellulose-like polysaccharides. Fruticose lichen thalli are composed of polysaccharides such as lichenin as well as chitin, and soluble carbohydrates but are low in protein and fiber. Lichenin is hydrolized to yield D-glucose and isolichenen is broken down to maltose (Llano, 1956). Rumen microbes detoxify lichen-secondary metabolites.

This exceptional arctic diet of lichens results in a unique rumen microbial community of 92.5% novel bacteria in Scandinavian reindeer (Sundset et al., 2007). Eubacterium rangiferina, a gram- negative, nonmotile rod bacterium was the first described capable of growth in usnic acid, known for its antimicrobial and toxic effects (Sundset et al., 2008). When thick snow or ice covers lichen mats, herders provide feed. Emaciated free-living reindeer fed fibrous hay or silage may succumb to starvation, regardless of feeding, because of failed digestion due to reduced essential ruminal microorganisms (Josefesen, 2007).

The effects of global warming climate on the growth and availability of lichens for reindeer lead to growing concerns. Recent decreases in lichen biodiversity will likely deleteriously impact reindeer and the Nomadic reindeer herder communities. Increasing ultraviolet radiation due to thinning of the ozone layer in the arctic may increase the synthesis of secondary metabolites in lichens as the compounds provide UV protection. Increases in toxicity of lichens may adversely affect reindeer (Sundset et al., 2010). Warmer climates may contribute to succession of vascular plants and a reduction of lichen mats (Cornelissen, 2004).

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Climate warming affects the geographical range suitable for reindeer. During the last glacial period, their range extended as far south as the northern reaches of Spain and in Mississippi in

North America (McDonald et al, 1996; García and Arsuaga, 2003). Reindeer populations can expand south during cool periods of glaciation and they retreat north during warmer periods.

Reindeer were extirpated from southern France by the end of the Pleistocene and an earlier temporary retreat from the Pyrenees to the Alps mountain ranges and through southern Europe corresponds to the Eemian interglacial period of global warming (Grayson and Delpech, 2005).

Reindeer fossil evidence in France along with summer climate data from paleobotany pollen counts shows reindeer populations decreased when global summer temperatures increased at the end of the last glaciation event (Grayson and Delpech, 2005). Recent climate warming and increased frequency of extreme weather events influence reindeer population dynamics, their current, synchronous population declines calls attention to the species' vulnerability to global change (Vors and Boyce, 2009). Loss of reindeer populations will deleteriously impact the circumpolar nomadic people who depend upon these exceptional ruminants.

6.9. Acknowledgements

I wish to express my appreciation to the University of Massachusetts Graduate School; to Dr.

Lynn Margulis and Dr. Richard Wilkie for instruction in Geosciences, focusing my attention on the importance of temporality and for the inspiration that initiated this project; and Jeremy

Sagan for computer technical support.

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Figure 6.1: The rumen symbio-organ of reindeer (Rangifera tarandus) harbors a community of symbiotic micro-organisms that are essential for the digestion of plants, lichens, and mushrooms

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Table 6.1: The symbiotic microorganisms required for the digestion of food in the rumen of free living reindeer Rangifer tarandus.

Protists:

19 regular species of ciliates found in free living Finnish reindeer (Westerling, 1970).

18 protist species found in reindeer from China, Russia, Finland, and Alaska (Imai et al., 2004).

Entodinium (9 species)

Diplodinium (2 species)

Eudiplodinium (2 species)

Ostrocodinium (3 species)

Enoplastron triloricatum

Epidinium ecaudatum

Dasytricha ruminatum

Anaerobic archae bacteria (methanogens):

(Sundset et. al., 2009; Margulis and Chapman, 2010)

Methanobacteriaceae (Methanobacteriales)

Methanobrevibacter spp. Is dominant in all ruminants.

Methanosarcinaceae (Methanosarcinales)

Methanomicrobiaceae (Methanomicrobiales)

Anaerobic eubacteria:

(Orpin et al., 1985;Cheng and McAllister, 1992;Aagnes et al., 1995, Sundset et. al., 2008)

Bacteroides

Fibrobacter

Streptococcus

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Enterococci

Clostridium

Clostridiales

Eubacterium rangiferina

Butyrivibrio fibrisolvens

Fusobacterium

Eubacterium

Ruminococcus

Enterobacteriacea

Anaerobic fungi [protoctist]:

(Gordon and Phillips, 1998; Sundset et. al., 2009; Margulis and Chapman, 2010)

Neocallimastigales

Chytridiomycete

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Table 6.2: Seasonal variation in ciliate (protist) and bacterial populations within the reindeer rumen and cecum microbial communities.

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6.10. References

Aagnes, T.H., Sørno, W., Mathiesen, S.D. 1995. Ruminal microbial digestion in free-living, in captive lichen-fed, and in starved reindeer (Rangifer tarandus tarandus) in winter.Appl Environ Microbiol. 61(2):583-91.

Aaris-Sørensen, K., Mühldorff, R., and Petersen, E. B.2007. The Scandinavian reindeer (Rangifer tarandus L.) after the last glacial maximum: time, seasonality and human exploitation. Journal of Archaeological Science. 34 (6): 914-923.

Annison, E.F. and Armstrong, D.G. 1970. Volatile fatty acid metabolism and energy supply. In: Phillipson, A. (Ed.), Physiology of Digestion and Metabolism in the Ruminant. Oriel Press, Newcastle upon Tyne, pp. 422-437.

Barnard, E.A. 1969. Biological function of pancreatic ribonucleause.Nature. 221:340-344.

Boertje, R. D. 1990. Diet quality and intake requirements of adult female caribou of the Denali herd, Alaska. Journal of Applied Ecology 27: 420-434.

Burch, E.,S. 1972. The Caribou/Wild Reindeer as a Human Resource.American Antiquity. 37:339- 368.

Cheng, K.J., McAllister T.A., Mathiesen, S.D., Blix, A.S., Orpin, C.G., Costerton, J.W. 1993. Seasonal changes in the adherent microflora of the rumen in high-arctic Svalbard reindeer. Can J Microbiol. 39(1):101-8.

Cheeke, P.R., and Dierenfeld, E.S. 2010. Comparative animal nutrition and metabolism. Cambridge University Press, Cambridge. Pp22-45.

124

Clauss, M., Hofmann, R.R. 2008. Higher masseter muscle mass in grazing than in browsing ruminants. Oecologia 157, 377-385.

Clauss, M., Hofmann, R.R., Fickel, J., Streich, W.J. and Hummel, J. 2009.The intraruminal papillation gradient in wild ruminants of different feeding types; implications for rumen physiology.Journal of Morphology. 270, 929-942.

Clauss, M., Kienzle, E. and Hatt, J.-M. 2003a. Feeding practice in captive wild ruminants: percularities in the nutrition of browsers/concentrate selectors and intermediate feeders. A review. In: Fidgettt, A., Clauss, M., Ganslosser, U., Hatt, J.-M. and Nijboer, J. (eds) Zoo animal Nutrition, vol. 2. Filander Verlag, Fürth, Germany, pp. 27-52.

Clauss, M., Lechner-Doll M., Streich, W.J. 2003. Ruminant diversification as an adaptation to the physiochemical characteristics of forage.A reevaluation of an old debate and a new hypothesis.Oikos. 102:253-262.

Colman, G.S. and Hall, F.J. 1984. The uptake and utilization of Entodiniium caudatum, bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa.Journal of Applied Bacteriology. 56(2):282-294.

Collinson, M.E. and Hooker, J.J. 1991. Fossil evidence of interactions between plants and plant- eating mammals.Philosophical Transactions of the Royal Society B. 333:197-200.

Cook, W.E., Raisbeck M.F., Cornish, T.E., Williams, E.S., Brown, B., Hiatt G., and Kreeger, T.J. 2007. Paresis and Death in Elk (Cervus elaphus) Due to Lichen Intoxication in Wyoming.Journal of Wildlife Diseases, 43(3): 498-503.

Decker, J.E., Pires, J.C., Conant, G.C., McKay, S.D., Heaton, M.P., Chen, K., Cooper, A., Vilkki, J., Seabury, C.M., Caetano, A.R., Johnson, G.S., Brenneman, R.A., Hanotte, O., Eggert, L.S., Wiener P, Kim JJ, Kim KS, Sonstegard TS, Van Tassell CP, Neibergs HL, McEwan JC, Brauning, R., Coutinho, L.L., Babar, M.E., Wilson, G.A., McClure, M.C., Rolf, M.M., Kim, J., Schnabel, R.D., Taylor, J.F. 2009. Resolving the evolution of extant and extinct ruminants with high-throughput phylogenomics.Proc Natl Acad Sci. 3;106(44):18644-9. 125

Dobson, D.E., Prager, E.M., Wilson, A.C. 1984. Stomach lysozymes of ruminants.International Journal of Biological Chemistry. 259:11607-11616.

Flagstad O., and Røed, K.H. 2003. Refugial origins of reindeer (Rangifer tarandus L.) inferred from Mitochondrial DNA sequences. Evolution. 57(3):658-70.

Gaare, E. 1995.A hypothesis to explain lichen-Rangifer dynamic relationships. Rangifer, 17 (1): 3-7.

García, N. and Arsuaga,J.L. 2003. Last glaciation cold-adapted faunas in the Iberian Peninsula. Deinsea 9:159-169.

Grayson, D.K. and Delpech, F. 2005. Pleistocene Reindeer and Global Warming.Conservation Biology. 19: 557-562.

Gripenberg, U., Wessman, M., Nieminen, M. 1986. The chromosomes of reindeer (Rangifer tarandus). Rangifer.1:109.

Gordon, G.L.R. and Phillips, M.W. 1998. The role of anaerobic gut fungi in ruminants. Nutrition Research Reviews. 11, 133-168.

Hackmann, T.J. and Spain, J.N. 2010. Invited review: ruminant ecology and evolution: perspectives useful to ruminant livestock research and production. Journal of Dairy Science. 93(4):1320-34.

Hobson, P.N., and Stewart, C.S. 1997. Rumen Microbial Ecosystem.2nd Edition.Springer.P. 567.

126

Hoffman, R.R., 1984. Comparative anatomical studies imply adaptive variations of ruminant digestive physiology. Canadian Journal of Animal Science 64, 203-205.

Hogg C, Neveu M, Stokkan KA, Folkow L, Cottrill P, Douglas R, Hunt DM, Jeffery G. 2011.

Arctic reindeer extend their visual range into the ultraviolet.Journal of Experimental Biology.214: 2014-9.

Holleman, D.F., Luick, J.R., White, R.G. 1979. Lichen intake estimates for reindeer and caribou during winter. The Journal of Wildlife Management, 43(1):192-201.

Hummel, M., and Justina C. R. 2008. Caribou and the North: A Shared Future. Toronto: Dundurn Press. Pp. 31-45.

Hungate, R.G. 1966. Rumen and its microbes. Academic Press, New York.

Imai, S., Oku, Y., Morita, T., Ike, K., Guirong. 2004. Rumen ciliate protozoal fauna of reindeer in Inner Mongolia. China. The Journal of Veterinary Medical Science.66(2):209-12.

Inga, B. 2007. Reinder (Rangifer tarandus tarandus) feeding on lichens and mushrooms: traditional ecological knowledge among reindeer-herding Sami in northern Sweeden. Rangifer, 27(2): 93-106.

Jernsletten, N. 1997. Sami Traditional Terminology. Professional Terms Concerning Salmon, Reindeer and Snow. In Harald Gaski, ed. Sami Culture in a New Era: The Norwegian Sami Experience. Kárásjohka, Norway. pp. 86-108.

127

Josefsen, T.D., Sørenson, K.K., Mørk, T., Mathiesen, S.D., Ryeng, K.A. 2007. Fatal inanition in reindeer (Rangifer tarandus tarandus): Pathological findings in completely emaciated carcasses. Acta Veterinaria Scandinavica. 49:27

Kalstad, J. K.H. 1997. “Aspects of Managing Renewable Resources in Sami Areas in Norway.” In Harald Gaski, ed. Sami Culture in a New Era: The Norwegian Sami Experience. Kárásjohka, Norway. pp. 109-126.

Klein D.R., Shulski, M. 2009. Lichen recovery following heavy grazing by reindeer delayed by climate warming. Ambio. 38 (1):11-16.

Llano, G. 1956.Utilization of Lichens in the Arctic and Subarctic. Economic Botany 10:367-392.

Mackie, R.I., Aminov, R.I., White, B.A., McSweeney, C.S. 2000. Molecular ecology and diversity in gut microbial ecosystems. In: Cronjé, P.B. (Ed.), Ruminant Physiology: Digestion, Metabolism, Growth and Reproduction. CAB International, New York, pp. 61-77.

Margolin, S. 1930. Methods for the cultivation of cattle ciliates.The Biological Bulletin. 59;301- 305.

Margulis, L., and Chapman, M.J. 2010. Kingdoms and Domains: An Illustrated Guide to the Phyla of Life on Earth, Elsevier/Academic Press.

Mathiesen, S.D., Rognmo, A., Blix, A.S. 1984.A test of the usefulness of a commercially available mill “waste product” (AB-84) as feed for starving reindeer. Rangifer 4, 28-34.

Mathiesen, S.D., Orpin, C.G., Greenwood, Y., Blix, A.S., 1987. Seasonal changes in the cecal micoflora of the high Arctic Svaldbard reindeer (Rangifer tarandus platyrhynchus). Appl. Environ. Microbiol. 53, 114-118.

128

Mathiesen, S.D. Sørmo, W., Aagnes-Utsi, T.H. 1999.Comparative aspects of volatile fatty acids in reindeer (Rangifer tarandus tarandus) in northern Norway and on South Georgia. Rangifer 20, 201-210.

McDonald, J.N., Ray, C.E., Grady, E. 1996. Pleistocene caribou (Rangifer tarandus) in the eastern United States: New records and range extensions. In K.M. Stewart and K.L. Seymour, (eds). Palaeoecology and paleoenvironments of the late Cenozoic mammals; tributes to the career of C.S. (Rufus) Churcher. University of Toronto Press, Toronto. Pp 406-430.

Moravi, D.P., Sakurada, M., Tomita,Y., Onodera R. 1996. Electrophoretic forms of chitinolytic and lysozyme activities in ruminal protozoa. Current Microbiology. 32(3): 115-118.

Newbold, C.J., Ushida, K., Morvan, B., Fonty, G., Jouan, J.P. 1996.The role of ciliate protozoa in the lysis of methanogenic archaea in rumen fluid.Letters in Applied Microbiology. 23(6);421-425.

Niemi, Einar. 1997. Sami History and the Frontier Myth.A Perpective on Northern Sami Spatial and Rights History. In Harald Gaski, ed. Sami Culture in a New Era: The Norwegian Sami Experience. Kárásjohka, Norway. pp. 62-85.

Olsen, M.A. 2000. Microbial digestion in reindeer and minke whatles.D. Phil. Thesis, University of Tromsø, Norway.p. 114.

Olsen, M.A. and Matheisen, S.D. 1998. The bacterial population adherent to plant particles in the rumen of reindeer (Rangifer tarandus tarandus) fed lichen, timothy hay or silage. Rangifer 18, 55-64.

Orpin, C.G., Mathiesen, S.D., Greenwood, Y., Blix, AS. 1985. Seasonal changes in the ruminal microflora of the high-arctic Svalbard reindeer (Rangifer tarandus platyrhynchus). Applied Environmental Microbiology. 50(1):144-51.

129

Randi, E., Mucci, N., Pierpaoli, M., Douzer, E. 1998. New phylogenetic perspectives on the Cervidae(Artiodactyla) are provided by the mitochondrialcytochrome b gene. Proceedings of the Royal Society of London B 265: 793-80.

Røed, K. H. 2005. Refugial origin and postglacial colonization of holartic reindeer and caribou. Rangifer, 25 (1) 19-30.

Sommer, R. S. and Nadachowski A. 2006. "Glacial refugia of mammals in Europe: evidence from fossil records". Mammal Rev 36 (4): 251–265.

Sørmo, W., Haga, Ø.E. Gaare, E., Langvatn, R., Mathiesen, S.D., 1999. Forage chemistry and digestive system in Svalbard Reindeer. Journal of Zoology. 251: 247-256.

Sundset, M.A., Praesteng, K.E., Cann, I.K., Mathiesen, S.D., Mackie R,I. 2007. Novel rumen bacterial diversity in two geographically separated sub-species of reindeer. Microbial Ecology. 54(3):424-38.

Sundset, M.A., Kohn, A., Mathiesen, S.D., Praesteng K.E. 2008.Eubacterium rangiferina, a novel usnic acid-resistant bacterium from the reindeer rumen.Naturwissenschaften. 95(8):741-9.

Sundset M.A., Edwards J.E., Cheng Y.F., Senosiain, R.S., Fraile, M.N., Northwood, K.S., Praesteng, K.E., Glad, T., Mathiesen, S.D., Wright, A.D. 2009. Rumen microbial diversity in Svalbard reindeer, with particular emphasis on methanogenic archaea. Microbial Ecology. 70(3):553-62.

Sundset M.A., Edwards J.E., Cheng Y.F., Senosiain, R.S., Fraile, M.N., Northwood, K.S., Praesteng, K.E., Glad, T., Mathiesen, S.D., Wright, A.D. 2009. Molecular Diversity of the Rumen Microbiome of Norwegian Reindeer on Natural Summer Pasture. Microbial Ecology; 57(2): 335-348.

Sundset, M.A., Barboza, P.S., Green, T.K., Folkow, L.P., Blix, A.S., Mathiesen, S.D. 2010. Microbial degradation of usnic acid in the reindeer rumen. Naturwissenschaften. 97(3):273-8.

130

Thomasson, J.R. and Voorhies, M.R. 1990. Grasslands and grazers. In D.E.G. Briggs and P.R. Crowther (eds.), Paleobiology: A synthesis. Blackwell Scientific Publ. Oxford. Pp. 84-87.

Vogels, G.D., Hoppe, W.F., Stumm, C.K. 1980. Association of methanogenic bacteria with rumen ciliates. Applied and Environmental Microbiology. 40(3): 608–612.

Vors, L. S. and Boyce, M. S.. 2009. Global declines of caribou and reindeer. Global Change Biology, 15: 2626–2633

Westerling, B. 1971. Rumen Ciliate Fauna of Semi-Domestic Reindeer (Rangifer Tarandus L.) in Finland: Composition, Volume and Some Seasonal Variations. Acta Zoologica Fennica 127: 76.

Western, D. 1975. Water availability and its influence on the structure and dynamics of a savannah large mammal community. East African Wildlife Journal, 13: 265-286.

Williams, A.G. and Coleman, G.S., 1988. The rumen protozoa. In: Hobson, P.N. (Ed.), The Rumen Microbial Ecosystem. Elsevier, London, pp. 77-128.

131

GENERAL BIBLIOGRAPHY

Aagnes, T.H., Sørno, W., Mathiesen, S.D. 1995. Ruminal microbial digestion in free- living, in captive lichen-fed, and in starved reindeer (Rangifer tarandus tarandus) in winter.Appl Environ Microbiol. 61(2):583-91.

Albignac, R., Rumpler, Y. and Petter, J. J. 1971. L’hybridation des lemuriens de Madagascar. Mammalia 35:358–368.

Awa A.A., Neel J.V. (1986) Cytogenetic “rogue” cells: What is their frequency, origin, and evolutionary significance? Proceedings of the National Academy of Sciences 83: 1021- 1025.

Barnard, E.A. 1969. Biological function of pancreatic ribonucleause.Nature. 221:340- 344.

Bantock C.R., Cockayne W.C. (1975) Chromosomal polymorphism in Nucella lapillus. Heredity. 34 (2):231-45.

132

Bonaccorso, F.J. 1998. Conservation International Tropical Field Guide Series: Bats of Papua New Guinea. Conservation International, Washington, D.C.

Burt G, Hauffe HC, Searle JB. 2009. New metacentric population of the house mouse (Mus musculus domesticus) found in Valchiavenna, Northern Italy. Cytogenet Genome Res. 125(4):260-5.

Blackburn E.H. (1991) Telomeres. Trends Biochem Sci. 16(10):378-81.

Bloom, K., Hill, A., Kenna, M. and Saunders, M. 1989.The structure of a primitive kinetochore. Trends Biochem.Sci. 14, 233–227.

Blower M. D., Sullivan B. A. and Karpen, G.,H. 2002. Conserved organization of centromeric chromatin in flies and humans. Dev. Cell. 2:319–330.

Boertje, R. D. 1990. Diet quality and intake requirements of adult female caribou of the Denali herd, Alaska. Journal of Applied Ecology 27: 420-434.

Bogart, M.H., Fujita, N., Serles, L., Hsia, Y.E. 1995. Prenatal diagnosis of a stable de novo centric fission: a case report. Am J Med Genet 23;59(1):36-37.

Brock J.K., Bloom, K. 1994. A chromosome breakage assay to monitor mitotic forces in budding yeast. Journal of Cell Science. 107; 891-902.

Burch, E.,S. 1972. The Caribou/Wild Reindeer as a Human Resource.American Antiquity. 37:339-368.

Chu, E. H. Y. 1975. Hapalemur griseus. In: An atlas of mammalian chromosomes. Hsu, T. C. and Binirscheke, Kl, ed., 9:441.

Chu, E. H. Y. and Swomley, B. A. 1961.Chromosomes of lemurine lemurs. Science 133:1925–1926.

133

Cheeke, P.R., and Dierenfeld, E.S. 2010. Comparative animal nutrition and metabolism. Cambridge University Press, Cambridge. Pp22-45.

Clauss, M., Hofmann, R.R. 2008. Higher masseter muscle mass in grazing than in browsing ruminants. Oecologia. 157, 377-385.

Clauss, M., Lechner-Doll M., Streich, W.J. 2003.Ruminant diversification as an adaptation to the physiochemical characteristics of forage.A reevaluation of an old debate and a new hypothesis. Oikos. 102:253-262.

Collinson, M.E. and Hooker, J.J. 1991. Fossil evidence of interactions between plants and plant-eating mammals. Philosophical Transactions of the Royal Society B. 333:197- 200.

134

DelPero, M., L. Pozzi, and J. C. Masters.2006. “A composite molecular phylogeny of living Lemuroid primates.” Folia Primatologica. 77:434-445.

Djlelati, R., Brun, B. and Rumpler, Y. 1997. Meiotic study of hybrids in the genus Eulemur and taxonomic considerations. Am. J. Primatol. 42:235–245.

Dobson, D.E., Prager, E.M., Wilson, A.C. 1984. Stomach lysozymes of ruminants.International Journal of Biological Chemistry. 259:11607-11616.

Dolan, M.F. 2005. The missing piece: the microtubule cytoskeleton and the origin of eukaryotes, in Microbial Phylogeny and Evolution: Concepts & Controversies. Sapp, J., ed.

Dolan, M.F., Melinitsky, H., Margulis, L., Kolnicki, R. 2002. Motility Proteins and the origin of the nucleus. The Anatomical Record 268:290-301.

135

Dutrillaux, B. and Y. Rumpler. 1977. Chromosomal evolution in Malagasy lemurs II. Meiosis in intra and interspecific hybrids in the genus Lemur. Cytogenetics and Cell Genetics 18:197-211.

Earnshaw , W.C., Tomkeil , J.E. 1992. Centromere and kinetochore structure. Current Opinion in Cell Biology 4: 86-83.

Earnshaw, W.C., Ratrie, H 3d, Stetten G. 1989. Visualization of centromere proteins CENP-B and CENP-C on a stable dicentric chromosome in cytological spreads. Chromosoma 98(1):1-12.

Flagstad O., and Røed, K.H. 2003. Refugial origins of reindeer (Rangifer tarandus L.) inferred from Mitochondrial DNA sequences. Evolution. 57(3):658-70.

Gaare, E. 1995. A hypothesis to explain lichen-Rangifer dynamic relationships. Rangifer, 17 (1): 3-7.

García, N. and Arsuaga,J.L. 2003. Last glaciation cold-adapted faunas in the Iberian Peninsula. Deinsea 9:159-169.

Gordon, G.L.R. and Phillips, M.W. 1998. The role of anaerobic gut fungi in ruminants. Nutrition Research Reviews. 11, 133-168.

Godfrey, L.R. and J. Masters. 2000. Kinetochore reproduction theory may explain rapid chromosome evolution. Proceedings of the National Academy of Sciences. 97:9821– 9823.

136

Gorbsky G.J., Sammak, P.J., Borisy, G.G. 1987. Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends. J. Cell Biol. 104:9-18.

Gorbsky G.J., Sammak, P.J., Borisy, G.G. 1988. Microtubule dynamics and chromosome motion visualized in living anaphase cells. J. Cell Biol. 106:1185-1192.

Grayson, D.K. and Delpech, F. 2005. Pleistocene Reindeer and Global Warming.Conservation Biology. 19: 557-562.

Greenbaum I.F., and M.J. Reed. 1984. Evidence for heterosynaptic pairing of the inverted segment in pericentric heterozygotes of the deer mouse (Peromyscus maniculatus). Cytogenetics and Cell Genetics38(2):160-111.

Gripenberg, U., Wessman, M., Nieminen, M. 1986. The chromosomes of reindeer (Rangifer tarandus). Rangifer.1:109.

Groves, C.P. 2000. The genus Cheirogaleus: Unrecognized biodiversity in dwarf lemurs. Int. J. Primatol. 21: 943–962.

Groves, C.P. and I. Tattersall. 1991. Geographical variation in the fork-marked lemur Phaner furcifer (Primates, Cheirogaleidae). Folia.Primatol. 56:39–49.

Guisto J. P. and Margulis, L. 1981. Karyotypic fission theory and the evolution of Old World monkeys and apes. Biosystems 13, 267-302.

Gutierrez J.M, Solorzano A, Cerdas L. 1984. Karyotype study of 5 species of Costa Rican snakes of the colubrid family. Rev Biol Trop. 32(2):263-7.

Haber, J.E. and Thorburn, P.C. 1984. Healing of broken linear dicentric chromosomes in yeast. Genetics 106(2):207-226.

Hamilton, A. E., Beuttner-Janusch, J. and Chu, E. H. 1977.Chromosomes of lemuriformes. II. Chromosome polymorphism in Lemur fulvus collaris (E. Geoffroy 1812). Am. J. Phys. Anthropol. 46:395–406.

137

Hackmann, T.J. and Spain, J.N. 2010. Invited review: ruminant ecology and evolution: perspectives useful to ruminant livestock research and production. Journal of Dairy Science. 93(4):1320-34.

Hill, A., Bloom, K. (1989) Acquisition and processing of a conditional dicentric chromosome in Saccharomyces cerevisiae. Mol Cell Biol 9(3):1368-70.

Hirai H., Yamamoto M.T., Taylor R.W., Imai H.T. (1996) Genomic dispersion of 28S rDNA during karyotypic evolution in the ant genus Myrmecia. Chromosoma 105(3):190-6.

Hobson, P.N., and Stewart, C.S. 1997. Rumen Microbial Ecosystem.2nd Edition.Springer.P. 567.

Hoffman, R.R., 1984. Comparative anatomical studies imply adaptive variations of ruminant digestive physiology. Canadian Journal of Animal Science 64, 203-205.

Hogg C, Neveu M, Stokkan KA, Folkow L, Cottrill P, Douglas R, Hunt DM, Jeffery G. 2011. Arctic reindeer extend their visual range into the ultraviolet.Journal of Experimental Biology. 214: 2014-9.

Holleman, D.F., Luick, J.R., White, R.G. 1979. Lichen intake estimates for reindeer and caribou during winter. The Journal of Wildlife Management, 43(1):192-201.

Hummel, M., and Justina C. R. 2008. Caribou and the North: A Shared Future. Toronto: Dundurn Press. Pp. 31-45.

Hungate, R.G. 1966. Rumen and its microbes. Academic Press, New York.

Hsu, T.C and Benirschke (1967-1975) An Atlas of Mammalian Chromosomes. Vols.1-9. Springer-Verlag, New York.

138

Imae, H. T., T. Maruyama, T. Gojori, Y. Inoue, and R. Crozier.1986. Theoretical bases for karyotype evolution. The minimum interaction hypotheses. American Naturalist 128: 900-920.

Imai H.T, Satta Y, Takahata N. 2001. Integrative study on chromosome evolution of mammals, ants and wasps based on the minimum interaction theory. J Theor Biol 21;210(4):475-97.

Imai, S., Oku, Y., Morita, T., Ike, K., Guirong. 2004. Rumen ciliate protozoal fauna of reindeer in Inner Mongolia. China. The Journal of Veterinary Medical Science.66(2):209- 12.

Inga, B. 2007. Reinder (Rangifer tarandus tarandus) feeding on lichens and mushrooms: traditional ecological knowledge among reindeer-herding Sami in northern Sweeden. Rangifer, 27(2): 93-106.

Joblonka E., and Lamb, M.J. 2006. Evolution in Four Dimensions: Genetic, Epigenetic, Behavioral, and Symbolic Variation in the History of Life. The MIT Press, Cambridge, MA.

Jager, D., Phillipson, P. 1989. Stabilization of dicentric chromosomes in Saccharomyces cerevisiae by telomere addition to broken ends or by centromere deletion. EMBO J. 8(1):247-254.

Jenkins, P.D. 1987. Catalogue of Primates in the British Museum (Natural History) and elsewhere in the British Isles. Part IV: Suborder Strepsirrhini, including the Subfossil Madagascan Lemurs and Family Tarsiidae. British Museum (Natural History), London.

139

King, Max. 1995. Species evolution: The role of chromosome change. Cambridge Univ Press, Melbourne Australia.

Klein D.R., Shulski, M. 2009. Lichen recovery following heavy grazing by reindeer delayed by climate warming. Ambio. 38 (1):11-16.

Kolnicki, R.L. 1999. Karyotypic fission theory applied - Kinetochore reproduction and lemur evolution. Symbiosis 26: 123-141.

Kolnicki, R.L. 2000. Kinetochore reproduction in animal evolution: Cell biological explanation of karyotypic fission theory. Proceedings of the National Academy of Sciences of the United States of America 97: 9493-9497. (Invited commentary by Godfrey, L.R., and Masters J.C. 2000 Kinetochore reproduction theory may explain rapid chromosome evolution. Proceedings of the National Academy of Sciences of the United States of America 97: 9821-9823.)

Kolnicki, R.L. 2009. Kinetochore reproduction (=karyotypic fission) theory: Divergence of lemur taxa subsequent to simple chromosomal mutation. (In review for Evolution)

Kolnicki, R.L. 2010. Lemurs and split chromosomes in Margulis, L. Asikainen C. and Krumbein, W.E., eds. Chimeras and Consciousness: Evolution of the Sensory Self (in press). Cambridge: MIT Press

140

Kolnicki, R. L. 2010 .Vespertilionidae (Chiroptera) chromosomal evolution explained by karyotypic fission theory (manuscript, work in progress).

Koopman, K.F. 1993. Chiroptera in Wilson, D.E., and D.M. Reeder, eds. Mammalian species of the World, 2nd ed., Smiths. Inst. Press, Washington, D.C., pp. 137-241.

Kuhn, T. 1996. The structure of scientific revolutions (3rded.). Chicago, IL: University of Chicago Press

Lamborot M. (1998) A new derived and highly polymorphic chromosomal race of monticola (Iguanidae) from the ‘Norte Chico’ of Chile. Chromosome Res. 6(4):247-54.

Llano, G. 1956. Utilization of Lichens in the Arctic and Subarctic. Economic Botany 10:367-392.

Margolin, S. 1930. Methods for the cultivation of cattle ciliates.The Biological Bulletin. 59;301-305.

Malik, H.S. and Henikoff, S. 2001. Adaptive evolution of cid, a centromere-specific histone in drosophila. Genetics 157:1293–1298.

Mantell, L.L., Greider, C.W. (1994) Telomerase activity in germline and embryonic cells of Xenopus. EMBO J. 1;13(13):3211-3217.

Margulis, Lynn. 1993. Symbiosis in Cell Evolution. San Francisco: W.H. Freeman, pp. 229- 272, 358-363.

141

Margulis, L., and Chapman, M.J. 2010. Kingdoms and Domains: An Illustrated Guide to the Phyla of Life on Earth, Elsevier/Academic Press.

Margulis, L. and Sagan, D. (2002) Acquiring genomes: A theory of the origins of species. New York, NY: Perseus Books Group (Basic Books)

Mathiesen, S.D., Rognmo, A., Blix, A.S. 1984.A test of the usefulness of a commercially available mill “waste product” (AB-84) as feed for starving reindeer. Rangifer 4, 28-34.

Mathiesen, S.D. Sørmo, W., Aagnes-Utsi, T.H. 1999.Comparative aspects of volatile fatty acids in reindeer (Rangifer tarandus tarandus) in northern Norway and on South Georgia. Rangifer 20, 201-210.

Matthey, R. 1973. The chromosome formulae of eutherian mammals. In A.B. Chiarellia and E. Capanna, (eds.): Cytotaxonomy and vertebrate evolution. Academic Press, London. pp. 531-616.

McFee, A. F., and M.W. Banner. 1969. Inheritance of chromosome number in pigs. Journal of Reproduction and Fertility 18: 9-14.

142

Moravi, D.P., Sakurada, M., Tomita,Y., Onodera R. 1996. Electrophoretic forms of chitinolytic and lysozyme activities in ruminal protozoa. Current Microbiology. 32(3): 115-118.

Montefalcone, G., Tempesta, S., Rocchi, M. and Archidiacono, M. 1999.Centromere repositioning. Genome Res. 9:1184–1188.

Moses, M.J., Karatsis, P. and Hamilton, A. 1979.Synaptonemal complex analysis of heteromorphic trivalents in lemur hybrids. Chromosoma 70: 141–160.

Nevo, 1999.Mosaic Evolution of Subterranean Mammals: Regression, Progression, and Global Convergence.Oxford University Press.

Neuweiler, G. 2000. The Biology of Bats. New York, NY: Oxford University Press.

Newbold, C.J., Ushida, K., Morvan, B., Fonty, G., Jouan, J.P. 1996. The role of ciliate protozoa in the lysis of methanogenic archaea in rumen fluid. Letters in Applied Microbiology. 23(6);421-425.

Nicklas, B.R. 1989. The Motor for Poleward Chromosome Movement in Anaphase Is in or near the Kinetochore. J. Cell Biol., 109:2234-2255.

Nicklas, R.B., Campbell, M.S., Ward, S.C., Gorbsky, G.J. (1998) Tension-sensitive kinetochore phosphorylation in vitro. J Cell Sci, 111 (21):3189-96.

Nowak, R.M. 1994. Walker’s Bats of the World. Baltimore, MD: The John Hopkins University Press.

O’Brien, S.J., Menninger, J.C., Nash, W.G. 2006. Atlas of Mammalian Chromosomes.John Wiley & Sons, Inc. Hoboken, NJ.

143

Olsen, M.A. 2000. Microbial digestion in reindeer and minke whatles.D. Phil. Thesis, University of Tromsø, Norway. p. 114.

Olsen, M.A. and Matheisen, S.D. 1998. The bacterial population adherent to plant particles in the rumen of reindeer (Rangifer tarandus tarandus) fed lichen, timothy hay or silage. Rangifer 18, 55-64.

O’Neill, R. J., Eldridge, M. D., Toder, R., Ferguson-Smith, M. A., O’Brien, P. C. & Graves, J. A. (1999) Chromosome evolution in kangaroos (Marsupialia: Macropodidae): cross species chromosome painting between the tammar wallaby and rock walaby spp. With the 2n=22 ancestral macropodid karyotype. Genome, 42(3):525-30

Pardo-Manuel de Villena, F. and Sapienza, C. 2001. Female meiosis drives karyotypic evolution in mammals. Genetics 159:1179–1189.

Pascoe, P.L., Patton S.J., Critcher R., Dixon D.R. (1996) Robertsonian polymorphism in the marine gastropod, Nucella lapillus: advances in karyology using rDNA loci and NORs. Chromosoma 104(6):455-60.

Pastorini, J., U. Thalmann, and R. D. Martin.2003. A molecular approach tocomparative phylogeography of extant Malagasy lemurs. Proceedings of the National Academy of Sciences. 100:5879-5884.

Paterson, H. E., 1978. More evidence against speciation by reinforcement. South African Journal of Science 74:369-371.

144

Perry, J., H. Slater, and K. Choo.2004. “Centric fission – simple and complexmechanisms.” Chromosome Research. 12:627-640.

Petter, J.J., Albignac, R. and Rumpler, Y. 1977.Faune de Madagascar 44: Mammiferes Lemuriens (Primates Prosimiens) O.R.S.T.O.M./C.N.R.S., Paris, 474–482.

Prowse K.R., Greider C.W. (1995) Development and tissue-specific regulation of mouse telomerase and telomere length. Proceedings of the National Academy of Sciences. 23;92(11):4818-22.

Reed, K.M., Sites J.W., Greenbaum, I.F. (1992) Synapsis, recombination, and meiotic segregation in the mesquite lizard, Sceloporus grammicus, complex. II. Fission heteromorphism of the FM2 cytotype and evolution of chromosome 2. Cytogenetics and Cell Genetics, 61(1):46-54.

Reig, O. A. and N.O. Bianchi. 1969. The occurrence of an intermediate didelphid karyotype in the short-tailed opossum (genus Monodelphis). Experientia 25:1210-11.

Røed, K. H. 2005. Refugial origin and postglacial colonization of holartic reindeer and caribou. Rangifer, 25 (1) 19-30.

Rofe, R. and Hayman, D. L. 1985. G-banding evidence for a conserved complement in the Marsupialia. Cytogenetics and Cell Genetics, 39:40–50.

145

Rousselt J, Geri C, Hewitt G.M., Lemuenier F. (1998) The chromosomes of Diprion pini and D. similis (Hymenoptera: Diprionidae): implications for karyotype evolution. Heredity 81 (5):573-8.

Rumpler, Y. 1975. Chromosomal studies in systematics of Malagasy lemurs. In: Lemur Biology, Tattersol, I. and Sussman, R., eds. Plenum Press, New York. Pp. 25–40.

Rumpler, Y. and Albignac, R. 1973. Cytogenic study of the Intrudae Burnett 1828, a malagasy endemic Lemur’s family (International Congress of Anthropological and Ethnological Sciences–conference and workshop on comparative kariology of primates), Detroit and Chicago.

Rumpler, Y. and Albignac, R. 1975. Intraspecific chromosome variability in a lemur from the north of Madagascar: Lepilemur septentrionalis, species nova. Am. J. Phys. Anthropol. 42:425–429.

Rumpler, Y. and Albignac, R. 1978. Chromosome studies of the Lepilemur, and endemic Malagasy genus of lemurs: contribution of the cytogenetics to their taxonomy. J. Hum. Evol. 7:191–196.

Rumpler, Y. and Albignac, R. 1980. Cytogenetic study of the Indridae Bennet, 1832, an endemic Malagasy lemur family. In B. Chiarelli, A.L. Koen, and G. Ardito, eds.: Comparative Karyology of Primates (Mouton Publishers, New York).

Rumpler, Y. J. Couturier, S. Warter, and B.Dutrillaux. 1983. Chromosomal evolution in Malagasy lemurs VII. Phylogenetic relationships between Propithecus, Avahi (Indridae), Microcebus (Cheirogaleidae), and Lemur (Lemuridae). Cytogenetics and Cell Genetics 36:542-546.

Rumpler, Y. and B. Dutrillaux. 1976. Chromosomal evolution in Malagasy lemurs III. Chromosome banding studies in the genuses Lemur and Microcebus. Cytogenetics and Cell Genetics 17:268-281.

146

Rumpler, Y. and B. Dutrillaux. 1978. Chromosomal evolution in Malagasy lemurs I. Chromosome banding studies in the genus Hapalemur and the species Lemur catta. Cytogenetics and Cell Genetics 21:201-211.

Rumpler, Y. and B. Dutrillaux. 1979. Chromosomal evolution in Malagasy lemurs IV. Chromosome banding studies in the genuses Phaner, Varecia, Lemur, Microcebus, and Cheirogaleus. Cytogenetics and Cell Genetics 24: 224-232.

Rumpler, Y. and B. Dutrillaux. 1980. Chromosomal evolution in Malagasy lemurs V. Chromosomal Banding Studies of Lemur fulvus albifrons, Lemur rubriventer and Its Hybrids with Lemur fulvus fulvus. Folia primatologica. 33:253-261

Rumpler, Y. and Dutrillaux, B. 1990. .Chromosomal evolution and speciation in primates. Cell Biol. Rev. 23:1–136.

Rumpler, Y., B. Ishak, S. Warter, and B. Dutrillaux. 1985. Chromosomal evolution in Malagasy lemurs VIII. Chromosome banding studies of Lepilemur ruficaudatus, L. leucopus, and L. septentrionalis. Cytogenetics and Cell Genetics 39:194-199.

Rumpler, Y., B. Ischak, B. Dutrillaux, S. Warter, and J.Ratsirarson. 1986. Chromosomal evolution in Malagasy lemurs IX. Chromosomal banding studies of Lepilemur mustelinus, L. dorsalis, and L. edwardsi. Cytogenetics and Cell Genetics 42:164-168.

Rumpler, Y., Ravaoarimanana, B., Hauwy, M. and Warter, S. 2001. Cytogenetic arguments in favour of a taxonomic revision of Lepilemur septentrionalis. Folia Primatol. 72:308–315.

Rumpler, Y., Prosper, P., Hauwy, M., Rabarivola, C., Rakotoarisoa, G. and Dutrillaux, B. 2002. Chromosomal evolution of the Hapalemur griseus subspecies (Malagasy 147

Prosimian), including a new chromosomal polymorphic cytotype. Chromosome Res. 10:145–53.

Rumpler, Y., Warter, S., Hauwy, M., Randrianasolo, V. and Dutrillaux B. 1991. Cytogenetic study of Hapalemur aureus. Am. J. Phys. Anthropol. 86:81–4.

Rumpler, Y., S. Warter, C. Rabarivola, J. Petter, and B. Dutrillaux. 1990. Chromosomal evolution in Malagasy lemurs XII. Chromosomal banding study of Avahi laniger occidentalis (Syn: Lichanotus laniger occidentalis) and cytogenetic Data in Favour of Its Classification in a Species Apart--Avahi occidentalis. American Journal of Primatology 21:307-316.

Saffery, R., Earle, E., Irvine, D.V., Kalitis, P., Choo, K.H. (1999) Conservation of centromere protein in vertebrates. Chromosome Res. 7(4):261-5.

Saffery, R., Irvine, D.V., Griffiths, B., Kalitsis, P., Wordeman, L., Choo, K,H. (2000) Human centromeres and neocentromeres show identical distribution patterns of >20

148

functionally important kinetochore-associated proteins. Hum Mol Genet Jan;9(2):175- 185.

Sapp, J. 2009. The New Foundations of Evolution: On the Tree of Life, Oxford University Press.

Skibbens, R.V., Hieter, P. (1998) Kinetochores and the checkpoint mechanism that monitors for defects in the chromosome segregation machinery. Annu Rev Genet 32:307-37.

Sørmo, W., Haga, Ø.E. Gaare, E., Langvatn, R., Mathiesen, S.D., 1999. Forage chemistry and digestive system in Svalbard Reindeer. Journal of Zoology. 251: 247-256.

Somonte, R.V., Ramesh K.H., Verma R.S., (1997) Comparative mapping of human alphoid satellite DNA repeat sequences in great apes. Genetica 101(2):97-104.

Sommer, R. S. and Nadachowski A. 2006. "Glacial refugia of mammals in Europe: evidence from fossil records". Mammal Rev 36 (4): 251–265.

Stanyon R., Koehler U., Consigliere S. 2002. Chromosome painting reveals that galagos have highly derived karyotypes. Am J Phys Anthropol 117:319-26.

Sundset, M.A., Kohn, A., Mathiesen, S.D., Praesteng K.E. 2008.Eubacterium rangiferina, a novel usnic acid-resistant bacterium from the reindeer rumen.Naturwissenschaften. 95(8):741-9.

149

Sundset, M.A., Barboza, P.S., Green, T.K., Folkow, L.P., Blix, A.S., Mathiesen, S.D. 2010. Microbial degradation of usnic acid in the reindeer rumen. Naturwissenschaften. 97(3):273-8.

Tagle, D. A., Goodman, M., and Miller, D. A. 1990.Characterization of chromosomes and localization of the rDNA locus in the aye-aye (Daubentonia madagascariensis).Cytogenet.Cell Genet. 54:43–46.

Tattersall, I. 1993. Speciation and morphological differentiation in the genus Lemur. Species, Species Concepts, and Primate Evolution, ed. Kimbel, W. and Martin, L., Plenum Press, NY, Pp. 163–176.

Taylor, S., and Mckenon, F. (1997) Kinetochore localization of Murine Bub1 is required for normal mitotic timing and checkpoint response to spindle damage. Cell 89;727-735.

Thomasson, J.R. and Voorhies, M.R. 1990. Grasslands and grazers. In D.E.G. Briggs and P.R. Crowther (eds.), Paleobiology: A synthesis. Blackwell Scientific Publ. Oxford. Pp. 84-87.

Todd, N. B. 1967. A theory of karyotypic fissioning, genetic potentiation and eutherian evolution. Mammalian Chromosome Newsletter 8:268–279.

Todd, N.B. 1970. Karyotypic Fissioning and Canid Phylogeny. Journal of Theoretical Biology 26:445-480.

Todd, N. B. 1975. Chromosomal mechanisms in the evolution of artiodactyls. Paleobiology 1:175–188.

Todd, N.B. 1985. Significance of a diploid number of 20 in the peccary Catagonus wagneri. The Journal of Heredity, 76:310.

150

Todd, N.B. 1992. Mammalian Evolution: karyotypic Fission Theory. In Margulis, L. and L. Olendzenski (ed.s): Environmental Evolution. Massachusetts Institute of Technology. Pp. 275-298.

Todd, N. B. 2000. Kinetochore reproduction underlies karyotypic fission theory: Possible legacy of symbiogenesis in mammalian chromosome evolution. Symbiosis 29:319-327.

Todd, N. B. 2000. Mammalian evolution: Karyotypic fission theory. In Environemental Evolution: Effects of the Origin and Evolution of Life on Planet Earrth, ed. L. Margulis, C. Matthews, and A. Haselton, second edition. MIT Press.

Toder, R., O'Neill, R. J. and Graves, J. A. 1998.Chromosome painting in marsupials.I.L.A.R. J. 39:92–95.

Ventura, M., Archidiacono, N. and Rocchi, M. 2001.Centromere emergence in evolution. Genome Res. 11:595–599.

Villasante A, Méndez-Lago M, Abad JP, Montejo de Garcíni E. 2007. The birth of the centromere.Cell Cycle. Dec; 6(23):2872-6.

Vig B.K. 1995. The centromere: kinetochore complex. Southest Asian J Trop Med Pub Health 26 Suppl 1:68-76.

Vogels, G.D., Hoppe, W.F., Stumm, C.K. 1980. Association of methanogenic bacteria with rumen ciliates.Applied and Environmental Microbiology. 40(3): 608–612.

Vors, L. S. and Boyce, M. S. 2009. Global declines of caribou and reindeer. Global Change Biology, 15: 2626–2633

Waters, J.J., Chen, R.H., Murray, A.W., Gorbsky, G.J., Salmo,n E.D., Nicklas, R.B. 1999. Mad2 binding by phosphhorylated kintochores links error detection and checkpoint action in mitosis. Curr Biol 17;9(12):649-52.

Warter, S., Randrianasolo, G., Dutrillaux, B. and Rumpler, Y. 1987. Cytogenetic study of a new subspecies of Hapalemur griseus.Folia Primatol. 48: 50–55.

Warter, S. Hauwy, M., Dutrillaux., and Rumpler, Y. 2005. Application of molecular cytogenetics for chromosomal evolution of the Lemuriformes (Prosimians). Cytogenet Genome Res 108:197-203.

151

Westerling, B. 1971. Rumen Ciliate Fauna of Semi-Domestic Reindeer (Rangifer Tarandus L.) in Finland: Composition, Volume and Some Seasonal Variations. Acta Zoologica Fennica 127: 76.

Western, D. 1975. Water availability and its influence on the structure and dynamics of a savannah large mammal community. East African Wildlife Journal, 13: 265-286.

White, M.J.D. 1973. Animal species and evolution. Cambridge University Press,

Cambridge, UK.

White, M.J.D., 1978. Modes of speciation. W.H. Freeman and Company, San Francisco.

Wilkie, R.W. 1991. “New England: A Region of Convergence and Ecological Diversity” in Historical Atlas of Massachutestts. Ed.s R. Wilkie and J. Tager. The University of Massachusetts Press. Amerst. Overview chapter, pp 2-3.

Williams, A.G. and Coleman, G.S., 1988. The rumen protozoa. In: Hobson, P.N. (Ed.), The Rumen Microbial Ecosystem. Elsevier, London, pp. 77-128.

Wilson, E.B. 1925. The Cell in Development and Heredity. MacMillan NY.

Wright, W.E., Piatyszek, M.A., Rainey, W.E., Byrd, W., Shay, J.W. (1996) Telomerase activitity in human germline and embryonic tissues and cells. Dev Genet 18(2):173-179.

Wurster-Hill, D.H. 1973.Chromosomes of the lemuridae. J. Hum. Evol.2:259.

Wyner, Y. M., Johnson, S. E., Stumpf, R. M. and Desalle, R. 2002. Genetic assessment of a white-collared x red-fronted lemur hybrid zone at Andringitra, Madagascar. J. Primatol. 57:51–66.

Yang, S.S., Yeh, E., Salmon, E.D., Bloom, K., 1997. Identification of a mid-anaphase checkpoint in budding yeast. J Cell Biol 27;136(2):345-354.

152

Yoder, A. D., Cartmill, M., Ruvolo, M., Smith, K. and Vilgalys, R. 1996. Ancient single origin for Malagasy primates. Proceedings of the National Academy of Sciences. 93:5122–5126.

Yu, C.W., Borgaonkar, D.S. Bolling, D.R. 1978. Break points in human chromosomes. Hum Hered 28(3):210-225.

Zaramody, A. and Pastorini, J. 2001. Indications for hybridisation between red-fronted lemurs (Eulemur fulvus rufus) and mongoose lemurs (E. mongoz) in northwest Madagascar. Lemur News 6:28–31.

Zubaid, A., McCracken, G.F., Kunz, T.H. 2006. Functional and Evolutionary Ecology of Bats. New York, NY: Oxford University Press.

153