Vertebrate Clonality: Genetics, Ecology, and Evolution of Sexual Abstinence

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(Oxford Univ. Press, 2008)
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general types of cloning

• artificial (human-mediated) vs. natural
• individual pieces of DNA vs. whole organisms

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artificial cloning of pieces of DNA
in vivo (via rest. enz., since late 1960s)
in vitro (via PCR, since mid-1980s)
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artificial cloning of whole vertebrate animals
As early as 1952, embryologists had cloned
individual frogs-- at least to the tadpole
stage-- using nuclear transfer (NT) from cells of
embryos or tadpoles and in 1975, John Gurdon
cloned frogs using NT from skin cells of adult
frogs
PNAS 38455-463 (1952)

• DEVELOPMENTAL CAPACITY OF NUCLEI TRANSPLANTED
  FROM KERATINIZED SKIN CELLS OF ADULT FROGS
• J. Embryology Exptl. Morph. 3493-112 (1975)

Leopard Frog (Rana pipiens)
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artificial cloning of mammals
nuclear-transfer (NT) technique
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ca. 20 mammal species have been artificially
cloned by NT

• to date, these include
• mouse
• rat
• goat
• pig
• wolf
• domestic dog
• muflon
• African wildcat
• domestic cat
• water buffalo
• gaur
• rabbit
• horse
• mule
• ferret
• and humans (to blastocyst stage)

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artificial cloning in the context of genetic
engineering
2007
2004
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natural cloning always based on DNA replication
and mitosis
DNA replication
mitosis
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1) unisexual clonality (parthenogenesis and
related modes) (known in ca. 100
species or distinctive biotypes) 2)
polyembryony (or monozygotic twinning)
(only one vertebrate species does this
constitutively)3) self-fertilization via
hermaphroditism (only one known
vertebrate species does this habitually)

• three forms of whole-animal vertebrate clonality
  occur in nature

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parthenogenesis reproduction by the chaste
parthenos means virgin, but ancient Athenians
worshipped the goddess Athena as the mother of
all men
(a sperm-independent and male-independent form of
all-female clonality)
replica of Athena Parthenos (original was
sculpted by Pheidias in 500 B.C., and housed in
the Parthenon)
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constitutive parthenogens cast of players
Darevskia rock lizards (ca. 7 different biotypes
or species)
Aspidoscelis (formerly Cnemidophorus) whiptail
lizards (gt 10)
Kentropyx whiptail lizards (1)
Gymnophthalmus spectacled lizards (1)
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constitutive parthenogens cast of players (cont.)
Lepidodactylus geckos (1)
Nactus geckos (1)
Heteronotia geckos (1)
Hemidactylus geckos (2)
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constitutive parthenogens cast of players (cont.)
Menetia skinks (1)
Leposoma spectacled lizards (1)
Ramphotyphlops blind snakes (1)
Lepidophyma night lizards (1)
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sporadic parthenogens some known examples
Komodo Dragon (Varanus komodoensis)
Bonnethead Shark (Sphyrna tiburo)
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gynogenesis hybridogenesis reproduction by
the semi-chaste
gynogenesis
hybridogenesis

• sperm-dependent forms of clonality
• unisexuals must be sympatric with males of sperm
  donor species
• males of those related species are sexually
  parasitized
• under hybridogenesis, males can be genetic
  fathers but not grandfathers

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gynogens and hybridogens cast of players
Poecilia mollies (gynogens)
Menidia silversides (gynogens)
Poeciliopsis livebearers (several diploid
hybridogens and triploid gynogens)
Carassius carps
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gynogens and hybridogens cast of players (cont.)
Phoxinus daces (gynogens)
Leuciscus minnows (hybridogens)
Fundulus killifishes (gynogens)
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gynogens and hybridogens cast of players (cont.)
Ambystoma mole salamanders (kleptogens)
Cobitis spined loaches (gynogens mostly)
Rana frogs (hybridogens)
Misgurnus loaches (gynogens)
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unisexual modes of clonality or hemiclonality
(sperm-independent)
(sperm-dependent)
(also KLEPTOGENESIS)
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geographic ranges and ecological success
Aspidoscelis (whiptail lizards)
Heteronotia binoei (Bynoes Gecko)
Darevskia (rock lizards)
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unisexual clonality background facts

• all known vertebrate unisexual species or
  biotypes originated via inter-specific
  hybridization
• each clonal line is thus highly heterozygous at
  nuclear loci
• many unisexual biotypes are diploid, but many
  others (ca. 60) are polyploid (usually triploid)

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what additional has been learned about unisexual
vertebrates from molecular markers (esp. from
cytonuclear analyses)?

• 1) the parent sexual species, and direction(s) of
  original hybrid cross, that produced each
  unisexual biotype
• 2) the cytogenetic mode by which clonal biotypes
  arose
• 3) the number of clonal strains within each
  biotype, their phylogenies, and their
  evolutionary ages

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• 1) the parent sexual species, and the
  direction(s) of original hybrid cross, that
  produced each unisexual strain

In 1978, M.J.D. White lamented in print that for
parthenogenetic species, we are never likely to
know which species was the female parent. But,
in 1979, Brown and Wright used mtDNA to document
the female parent in two Aspidoscelis
(Cnemidophorus) parthenogens. Since then,
cytonuclear appraisals have been employed to
reveal, unambiguously, the male and the female
parent species for many of the worlds known
unisexual biotypes
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the logic of cytonuclear analysis is
straightforward in this context

• nl1 nl2 nl3 nl4 nl5 nl6 mt
• candidate sexual parents
• sp1 A C D B A D A
• sp2 A,B F D,E C A B B
• sp3 A,C D,F A,C D B A,D C
• sp4 B,C A B A,B D B,D D
• sp5 D,E C C A,C E A,D E
• parthenogenetic lineage A/E C/F C/D
  C A/E B/D E
• so, in this case, the parthenogenetic biotype
  clearly arose via a cross
• between a female from sp5 and a male from sp2

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example Amazon Molly, Poecilia formosa (a
gynogenetic species that sexually parasitizes
males of sympatric sexual species, normally
either P. mexicana or P. latipinna)

• molecular markers (mtDNA and allozymes) confirmed
  that the Amazon Molly arose recently in evolution
  from a hybridization event between a P. mexicana
  female and a P. latipinna male.

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unisexual biotype mode male parent
female parentAspidoscelis lizards
uniparens parthenogen burti
inornatus tesselatus
parthenogen septemvittatus marmoratus
velox parthenogen
inornatus burti or costatus
laredoensis parthenogen sexlineatus
gularis Heteronotia
binoei-type lizards parthenogen binoei
sp. CA6 Menidia fish
clarkhubbsi complex gynogen
beryllina peninsulae Phoxinus
fish eos-neogaeus gynogen
eos neogaeus Poeciliopsis fish
monacha-lucida hybridogen
lucida monacha
monacha-occidentalis hybridogen
occidentalis monacha
a few more such representative examples
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invariably, the bisexual parent species have
proved to be somewhat distant congeners (i.e.,
not sister taxa)
example Darevskia lizards
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more such examples
Aspidoscelis lizards
Poeciliopsis fishes
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why are the clone-yielding hybridization events
between non-sister species?its not because
sister species cant hybridize they often do
hybridize, but the hybrids are sexual rather than
parthenogenetic

• two conventional hypotheses (not mutually
  exclusive)
• balance hypothesis (Moritz, Vrijenhoek)
• posits that unisexuals arise only when the
  genomes of the parental species are divergent
  enough to disrupt meiosis in hybrids yet no so
  divergent as to seriously compromise hybrid
  viability or fertility
• phylogenetic constraint hypothesis (Darevsky)
• posits that genetic peculiarities happen to
  predispose particular parental species to produce
  unisexual lineages when they hybridize

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• 2) the cytogenetic mode by which triploid
  clonal strains arose

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two hypothetical routes to triploid unisexuals
1) primary-hybrid route the paired homospecific
nuclear genomes should derive from the sexual
species that provided the male parent in the
original hybrid cross 2) spontaneous-origin
route the paired homospecific nuclear genomes
should derive from the sexual species that
provided the female parent (and mtDNA) in the
original hybrid cross
in empirical cytonuclear appraisals, the
primary-hybrid route proved correct for most if
not all Aspidoscelis lizards examined also true
for triploid Poeciliopsis unisexuals
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• 3) the number of clonal strains, their
  phylogenies, and their evolutionary ages
• most of this information has come primarily from
  analyses of mtDNA (molecular clones within the
  organismal clones)

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origin of a unisexual lineage
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example of mtDNA phylogeny (monophyly in this
case)
Aspidoscelis (whiptail lizards)
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another example of mtDNA monophyly for a
unisexual taxon
Ambystoma (mole salamanders)
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examples of mtDNA polyphyly for a unisexual taxon
Heteronotia binoei (Bynoes Gecko)
Menetia greyii (Dwarf Skink)
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estimated evolutionary ages of unisexual
taxa(based on observed mtDNA divergence between
each unisexual lineage and its closest extant
maternal ancestor)
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mtDNA nucleotide diversities in maternal bisexual
species are usually much higher than those in the
unisexual biotypes to which they respectively
gave rise
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1) unisexual clonality (parthenogenesis and
related modes) (known in ca. 100
species or distinctive biotypes) 2)
polyembryony (or monozygotic twinning)
(only one vertebrate species does this
constitutively)3) self-fertilization via
hermaphroditism (only one known
vertebrate species does this habitually)

• three forms of whole-animal vertebrate clonality
  occur in nature

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sporadic polyembryony some reported examples

• cattle
• pigs
• deer
• whales
• various avian species
• some fish species
• and, of course, humans (monozygotic twins)

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constitutivepolyembryony
Dasypus novemcinctus (nine-banded armadillo)

• armadillos (Dasypus) seem to be unique among
  vertebrates in this peculiar reproductive
  phenomenon.
• an intra-generational rather than
  inter-generational form of clonality
• are littermates truly polyembryonic?
• how might polyembryony have evolved?

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microsatellite confirmation of polyembryony
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Dasypus armadillos
parasitoid wasps
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1) unisexual clonality (parthenogenesis and
related modes) (known in ca. 100
species or distinctive biotypes) 2)
polyembryony or twinning (only one
vertebrate species does this constitutively)3)
self-fertilization via hermaphroditism
(one known vertebrate species does this
habitually)

• three forms of whole-animal vertebrate clonality
  occur in nature

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mangrove killifish the fish that mates with
itself

• Kryptolebias (formerly Rivulus) marmoratus is the
  worlds only known vertebrate species that
  regularly reproduces as a self-fertilizing
  hermaphrodite.
• Inside each fishs body is an ovotestis that
  simultaneously produces eggs and sperm. These
  gametes normally unite inside the fish, who then
  lays the self-fertilized eggs or zygotes
  externally, where embryonic development proceeds.
• Self-fertilization is a highly intense form of
  inbreeding. It seems to have produced highly
  homozygous lineages of K. marmoratus whose
  members are so genetically uniform that they
  traditionally have been referred to as belonging
  to clones (as gauged, for example, by
  acceptance of skin grafts).

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selfing and heterozygosity decay
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27 variable microsatellite loci
Individuals from local Florida sites
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27 variable microsatellite loci
Individuals from local Florida sites
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our genetic findings to date

• K. marmoratus is really an androdioecious species
  whose local populations consist of mixtures of
  simultaneous hermaphrodites and pure males
• selfing predominates, but occasional outcross
  events (probably mediated by males) release local
  bursts of multi-locus heterozygosity available
  for subsequent Mendelian segregation and
  reassortment into a wide variety of
  recombinant-inbred (and highly homozygous)
  strains
• local populations thus have a mixed-mating system
  (predominant selfing with occasional outcrossing)
• this mixed-mating system and its resulting
  population-genetic architecture in the Mangrove
  Killifish provide a remarkable case of convergent
  evolution on the mating systems and population
  genetic patterns previously reported in otherwise
  very different kinds of organisms
• some annual plants (e.g., the slender wild
  oat)
• some invertebrate animals (e.g., various land
  snails)

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heterozygosity issues(and Bakers rule too)
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the end
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given the primary-hybrid route, there are two
further possible hypotheses for triploid
unisexuals

• genome-addition hypothesis
• unreduced ova from the F1 hybrid carried genomes
  from both of the parental species
• genome-duplication hypothesis
• unreduced ova from the F1 hybrid carried two
  copies of a genome from only one of the parental
  species

in empirical cytonuclear appraisals, the
genome-addition route proved to be true for
unisexual Poeciliopsis, Poecilia, Menidia, and
Phoxinus
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Part I. Background Like Begets Like
Chapter 1. Clonality within the Individual
Chapter 2. Sexuality the Antithesis of
ClonalityPart II. Unisexual Clonality in
Nature Chapter 3. Reproduction by the
Chaste Parthenogenesis Chapter 4.
Reproduction by the Semi-chaste Gynogenesis,
Hybridogenesis, and KleptogenesisPart III.
Sexual Clonality in Nature Chapter 5.
Clonality In Utero Polyembryony Chapter 6.
Clonality by Incest Hermaphroditic
Self- fertilizationPart IV. Clonality in the
Laboratory Chapter 7. Human-sponsored
Clonality
(chapter outline)
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a 100,000-year-old hemiclone in Poeciliopsis

• unisexual lineages (black circles in the mtDNA
  network) of Poeciliopsis have arisen many times
  in evolution.
• Indeed, hybridogenetic strains can be lab-
  produced by crossing related sexual species.
• most of the lineages in nature appear to be
  evolutionarily recent or modern
• however, based in part on a moderate level of
  (post-formational) genetic variation in one
  strain that from multiple lines of evidence is
  monophyletic, we (Quattro et al.) estimated that
  this lineage is approximately 100,000 generations
  old.

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the line-curves are the expected heterozygosities
under.

• random mating
• random mating
• mixed mating
• (with s 0.44
• and
• t 0.56)

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challenges in estimating the number of hybrid
geneses (and evol. ages) for unisexual biotypes

• face-value taxonomy may be misleading, in either
  direction (various unisexual taxa might be
  oversplit, or overlumped, with respect to the
  number of independent evolutionary origins)
• the difficulty of properly apportioning any
  genetic differences observed among extant
  unisexuals to independent geneses versus
  post-formational mutations
• the need to appreciate that a unique genesis for
  mtDNA in a unisexual taxon does not necessarily
  equate to a single hybrid origin
• (imagine, for example, that an Eve had two or
  more mates then, the unisexual biotype would be
  monophyletic for mtDNA, and yet show considerable
  nuclear genome diversity)
• the problem of missing data due to extinction
• (for example, the sexual ancestor of a unisexual
  biotype may have gone extinct)
• the many caveats about molecular clocks

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outcrossing in Belize
Twin Cays, Belize
Charlotte Co., FL.
Shark River, FL.