Section 13Use of molecular genetics in forensics &

to understand species biology

Genetic markers contribute to the conservationof species by aiding in detection of illegalhunting and by resolving important aspects ofspecies biology; genetic markers have been used for the following:

To detect bottlenecks & other demographicevents in a populations history

Estimating effective population size

Detecting selection

Determining parentage, gender, mating systems,population structure, dispersal rates, population Size, diet, and disease status

Poaching and illegal harvest are threats to a widevariety of endangered species, especially largecats, elephants, bears, rhinos,parrots, whales,and some plants.

Most countries have laws to protect threatenedplants and animals.

However, it is often difficult to obtain evidenceto convict individuals suspected of illegally takingor trading in threatened species -- case of case of Australian airport, person, eggsAustralian airport, person, eggs.

Molecular genetic techniques have assumed animportant and growing role in the detection ofillegal hunting of wildlife.

The US Fish and Wildlife Service has establishedThe Clark Bevin Forensics Laboratory in Oregonspecifically for the purposes of providingevidence in cases involving illegal exports, imports & hunting of endangered species.

Following many years of commercial exploitation,The numbers of most whale species collapsed.

This led the International Whaling Commission(IWC) to institute a global moratorium onCommercial whaling that took effect in 1985/86

Some IWC members havecontinued to hunt a fewwhale species (primarilyminke whales) for scientificpurposes and the whale meat can be sold for humanconsumption.

There were suspicions that protected whalespecies were being marketed as species thatcould be taken legally.

At the request of Earthtrust, Baker & Palumbideveloped a PCR based system for monitoringtrade in whale and dolphin products based on mtDNA sequence variation.

First, they reliably distinguished a variety of whale and dolphin species from each other usingmtDNA control region sequence variation.

Samples of whale products were subsequentlypurchased in retail markets in Japan and Korea.

Samples of whale products were subsequentlypurchased in retail markets in Japan and Korea.

Min

ke w

hale

Min

ke w

hale

Hum

pbac

k w

hale

Hum

pbac

k w

hale

Gra

y w

hale

Gra

y w

hale

Blu

e w

hale

Blu

e w

hale

Fin

wha

leFi

n w

hale

Sei w

hale

Sei w

hale

Bry

de’s

wha

leBow

head

wha

le

Bow

head

wha

le

Rig

ht w

hale

Pygm

y righ

t w

hale

Sper

m w

hale

Pygm

y sp

erm

wha

le

Har

bor

porp

oise

Hec

tor’

s do

lphi

n

Com

mer

son’

s do

lphi

n

Kille

r w

hale

To avoid the possibility of violating laws governingtransport of endangered species, Baker & Palumbiset up a portable PCR laboratory in their hotelroom and amplified the mtDNA control regionfrom the samples.

The amplified DNA was taken back to their labsin New Zealand and the USA and sequenced.

Following is their results from the original 16purchases:

Min

ke w

hale

Min

ke w

hale

Sam

ple

#19a

Sam

ple

WS

3S

am

ple

#9

Sam

ple

#15

Sam

ple

#29

Sam

ple

#30

Sam

ple

#36

Sam

ple

#6

Min

ke w

hale

Sam

ple

#18

Sam

ple

#19b

Hum

pback

whale

Hum

pback

whale

Gra

y w

hale

Gra

y w

hale

Blu

e w

hale

Blu

e w

hale

Sam

ple

#41

Sam

ple

#3

Sam

ple

#11

Sam

ple

WS

4Fi

n w

hale

Fin

whale

Sei w

hale

Sei w

hale

Bry

de’s

whale

Bow

head w

hale

Bow

head w

hale

Rig

ht

whale

Pygm

y r

igh

t w

hale

Sp

erm

whale

Pygm

y s

perm

wh

ale

Sam

ple

#16

Harb

or

porp

ois

eS

am

ple

#13

Sam

ple

#28

Hect

or’

s dolp

hin

Com

mers

on’s

dolp

hin

Kill

er

whale

By 1999, 954 samples of “whale meat” had beenpurchased in Japan and Korea and analyzed byvarious scientific groups; 773 (81%) were fromwhales, approximately 9% coming from protectedwhale species.

Samples not from whales included dolphins,porpoises, sheep, and horses.

Not only were consumers being misled, but therewere questions regarding the origin of the meat.

The possibility that meat from protected specieshad been sourced from frozen stores collectedprior to bans on whaling cannot be excluded, butthis explanation does not apply to fresh meat.

This has led to stricter controls over the distribution of “scientifically harvested” whalemeat and demands that legally harvested whalesand meat stockpiled prior to whaling bans begenetically typed to monitor distribution.

Similar to the example with whales, a PCR-based mtDNA analysis revealed that 23% of caviarsamples in New York was mislabelled.

The sources of the caviar have conservationimplications, as most of the 27 members of thesturgeon group are endangered due to overfishingand habitat degredation.

The identity of a poached endangered Arabianoryx was confirmed by microsatellite analysesand mtDNA-based methods are being developedto detect tiger products in Asian medicines.

Forensic DNA methods have been used todetermine the source of poached chimpanzees.

Sequencing of mtDNA established that 26confiscated chimpanzees in Uganda belonged tothe eastern subspecies.

This identified the region where poaching wastaking place, and where these animals could bereinstated into the wild.

Gene Trees and Coalescence

Coalescence and gene trees derived from data onsequence differences among individuals andpopulations are important tools for exploringevolutionary processes and demographic eventsin a species’ past.

Based on neutral theory, these provide a nullhypothesis against which to test data and todiscriminate possible reasons for deviations.

Moreover, calescent methods work backwards intime and allow time dimensions (generations) to beadded to the analyses.

Consequently, they are more powerful than conventional analyses that use only currentdistributions and patterns of DNA sequencedifference.

Coalescence is based on the concept that currentallelic sequences in a population can be traced back through time to a point at which they coalesce to a single individual sequence.

Other alleles, once present in the past, have been lost by genetic drift or selection, and newalleles have been generated through mutation.

The evolutionary pattern of the extant distributionof alleles at a locus can be represented as thebranches of a tree coalescing back to a singleancestral allelic sequence.

Coalescent patterns are usually depicted usinggene trees, which show the genealogy of the allelesin the current population.

The nodes (coalescent events) and branch lengthsin the tree reflect the origins and time framesinvolved in deriving the observed patterns.

Gene trees trace the evolutionary history of allelesin the same manner as tracing the origin, or loss,of alleles through pedigrees.

A EDCB F

0

PAST

PRESENT

DIVERGENCE

A EDCB F

0

PAST

PRESENTCOALESCENCE

The basis of the coalescence method is that DNAsequence differences among alleles at a locus retain information about the evolutionary historyof those sequences.

For example, two alleles that differ by 2 basesare more closely related and diverged more recently than 2 alleles that differ by 11 base pairs.

Neutral theory allows us to predict the time ingenerations back to coalescence, thus adding a time dimension to analyses.

Under neutral theory, two alleles may descendfrom the same ancestral allele in the previousgeneration with a probability 1/Nef for mtDNA,or 1/2Ne for a nuclear diploid locus.

Alternatively, two alleles may derive from twodifferent alleles in the previous generation (orderived from the same allele many generationsago) with probabilities 1 - 1/Nef, or 1 - 1/2Ne.

This is the same reasoning used to determine lossof genetic diversity.

Under the neutral model of genetic drift, thecoalescence process takes a characteristic time.

In a diploid population with k alleles at a neutrallocus, the average time Tk back to the previouscoalescent event (i.e., where there were k - 1alleles) is:

Tk = 4Ne/[k(k-1)] generations.

1 2 3 4 5 T5 E(T5) = 2N/10

T4 E(T4) = 2N/6

T3 E(T3) = 2N/3

T2 E(T2) = 2NPast

Present

Thus, the timesduring which There are 5, 4, 3, and 2 lineages are 2Ne/10, 2Ne/6, 2Ne/3, and 2Ne generations, respectively.

The time for all alleles in the population to coalesce is 4Ne/[1 - (1/k)] generations.

Thus, the coalescence is quicker, and gene treesshorter, in smaller than larger populations.

Thus, it should be obvious to see that gene treeanalyses can provide information about differences in historical population size fordifferent populations or species.

Example: In a population with Ne = 50 with 3alleles, the expected time to its previous coalescence (when the population had only 2alleles) is:

T3 = 4Ne/[k(k-1)] = (4X50)/(3X2) = 33 generations

Thus, 3 alleles will coalesce to 2 alleles on averagein 33 generations in a population of size Ne = 50.

For Ne = 100 coalescence takes:

T3=4Ne/[k(k-1)] = (4X100)/(3X2) = 67 generations.

Thus, the coalescence takes twice as long in a population with twice the size.

Therefore, the coalescence times increase indirect proportion to population size.

The structure of gene trees and patterns ofcoalescence are strongly influenced by deviationsfrom neutrality and random mating.

For example, different forms of selection affectthe coalescence time in characteristics ways;directional selection reduces the coalescence time, while balancing selection increasescoalescence time, compared to the expectationwith genetic drift.

Neutral BalancingSelection

DirectionalSelection

After long periods of isolation and lack of geneflow, populations show deep divisions among them.

Migration yeilds characteristic signatures whengene trees are mapped onto geographic locations,alleles characteristic of one geographic regionare found in another, partially isolated, region.

Fluctuations in population size of populationbottlenecks foreshorten coalescence time.

Mutations generate sequence differences, slowingcoalescence times.

Neutral

Locality A Locality B

GeographicIsolation

Locality BLocality A

MigrationPopulationBottleneck

When patterns are Similar, such as thoseFor directional selectionAnd populationBottlenecks, additionalIniformation is requiredTo resolve the cause.

For example, information on multiple unlinked lociAllows discrimination of directional selection andBottlenecks; bottlenecks affect all loci in a similarManner while directional selection will affect eachLocus in a different manner.

PopulationBottleneck

DirectionalSelection

Differences in DNA sequences, gene tree structure and coalescence rates allow us to infer details about population structure andevolution that are not easily, or less acurately,found using other techniques.

Analysis of gene trees, using coalescence analysis, have been used to:

Estimate effective population size (usingselectively neutral markers)

Measure neutral mutatation ratesInfer selection and determine its formDetermine migration events and measure

migration ratesDetermine phylogenetic relationships among

geographically separated populationsDetect secondary contact of diverged pops.Estimate divergence times among pops.Infer changes in population sizesReconstruct the origins and history of disease

epidemics

Demographic History

The distribution of the number of sequencedifferences between pairs of alleles ( a “mismatch”analysis) has characteristic shapes for populationswith different demographic histories.

Populations with historically stable population size,exponentially growing populations, populationbottlenecks, or populations experiencing secondarycontact leave different signals.

Pairwise Differences

Frequency

Pairwise Differences

Frequency

Stable

Exponentiallygrowing

Stable population growthresults in a geometricdistribution.

Exponential growth isexpected to generatea smooth unimodaldistribution.

Pairwise Differences

Frequency

PopulationBottleneck

Pairwise Differences

Frequency

SecondaryContact

Bottlenecks yeild either adistribution close to zero,or a bimodal distribution, depending on whether thebottleneck reduced geneticdiversity, or completelyremoved it (so that diversityrepresents mutations sincethat point).

Humans exhibit a unimodal distributioncharacteristic of exponential growth, which accords with known human history.

Undocumented past bottlenecks can be detectedand their severity inferred from the loss ofgenetic diversity.

Even when there are no samples of the pre-bottleneck population, they can often beidentified using information from multiplemicrosatellite loci.

Reintroduction of koalas in southeastern Australia: a poorly designed program with adverse genetic impacts.

The koala is a unique marsupialendemic to eastern Australia.

It is both a cultural icon and animportant contributor to touristincome (~$70 million/yr).

The koala once ranged downthe East Coast fromQueensland to Victoria and South Australia but its numbers have been reduced by hunting, habitat loss, anddisease.

At the peak of hunting in 1924, 2 million animals were shot.

By the 1930s, koalas inhabited less than 50% of their former range.

They had disappeared in South Australia and were nearly extinct in Victoria.

However, they were still considered common inQueensland where they subsequently recoveredwithout large-scale assistance.

The fur trade ceased by the 1930s when koalaswere accorded legal protection in all states.

Subsequently, much effort has gone into koala conservation.

Extensive translocations of animals occurred in the southeast.

A population was founded from as few as 2 - 3individuals on French Island (FI) in Victoria latein the 19th century.

FI

This population grew rapidly inthe absence of predators andrapidly reached carryingcapacity.

Surplus individuals from thispopulation were used to found an additionalpopulation on Kangaroo Island (KI) (18 adult Founders plus young) in 1923 - 1925, and to supplement a population founded on Phillip Island(PI) in the 1870s.

FIKI

PI

FIKI

PI

The French and Phillip Islandpopulations were used widely tosupplement mainland SouthAustralia.

The restocked South Australiamainland population has gone through threebottlenecks: Mainland Victoria --> French Is. --> Kangaroo Is. --> mainland South Australia.

Since 1923, 10,000 individuals have been translocated to 70 locations.

Stocking of populations using individuals from bottlenecked populations is expected to result in loss of genetic diversity and inbreeding.

As predicted, the southeastern populations ofVictoria and South Australia have about halfthe genetic diversity found in the less-perturbedpopulations further north: 5.3 vs. 11.5 microsatellitealleles per locus and heterozygosities (He) of 0.44 vs. 0.85.

FIKI

PI

FIKI

PI

DNA fingerprint and RAPDanalyses provide similar results.

As expected, the Kangaroo Is.population had the lowest genetic diversity of all surveyedpopulations.

All southeast populations showed similarmicrosatellite allele frequencies, and similar mtDNAhaplotypes, while the more northern populationsexhibited considerable differentiation.

FI

KI

PI

BRSR

SGSZ

CBML

NCWNSW

IL

SP

GC

MT

TB

FIPI

KISRBRSG

NCCB

WNSWML

IL

MTGC

SP

Gene tree for koala populations, based on mtDNA sequence divergence. Populations from Victoria & South Australia (bottom), derived mainly from bottlenecked island populations, are essentially indistinguishable. The remaining populations generally show their closest affinities withgeographically adjacent populations, as expected with isolation by distance.

Thus, translocations using individuals with lowgenetic diversity have reduced genetic diversityand distorted natural allele frequencies.

The translocations may have contributed to reductions in reproductive fitness, includinglowered resistance to Chlamydia infection, to a lowering of sperm concentrations and motility,and to an increased frequency in testicularabnormality.

What might have been done to avoid such problems?

Loss of genetic diversity and inbreeding depressionwould have been averted if the French Islandpopulation had been founded with more individuals,or if its genetic diversity had been augmentedto give it a greater base of genetic diversity.

The situation would have been much better if themore diverse Phillip Island population had not been“swamped” with French Island individuals.

What can be done to reverse the problem?

The most efficient strategy would be to introducemore genetic diversity into the southeasternpopulations (both mainland and island).

Since the koala population is somewhat differentiated, this would best be done from nearby populations.

The ideal solution would be discovery of aremnant population in Victory with high geneticdiversity.

If none exist, then the best source of geneticdiversity is from New South Wales.

Genetic diversity should be checked in sourcepopulations before they are used for translocations.

Bottleneck effects have been measured bycomparing microsatellite genetic diversity fromthe current populations with that frommuseum speciemens as was done for Mauritiuskestrels.

The decline of the Mauritius kestrel began withthe destruction of native forest and the plungetowards extinction resulted from thinning of eggshells and greatly reduced hatchabilityfollowing use of DDT insecticides beginning inthe 1940s.

In 1974, its population numbered only fourindividuals, with the subsequent populationdescending from only a single breeding pair.

Under intensive management the population grewto 400 - 500 birds by 1997, but it experiencedsix generations at numbers less than 50.

While this is a success story, the Mauritius kestrelcarries genetic scars from its near extinction.

It now has a very low level of genetic diversity for12 microsatellite loci, compared to six other kestrel populations.

The Mauritius kestrel has 72% lower allelic diversity and 85% lower heterozygosity than the mean of the non-endangered kestrels.

Prior to its decline, the Mauritius kestrel hadsubstantial genetic diversity, based on ancestralmuseum skins from 1829 - 1894, but even then itsgenetic diversity was lower than the non-endangered species.

The Seychelles kestrel went through a paralleldecline and recovery and also has low genetic diversity.

The Seychelles kestrel was rare during the 1960sand had become extinct on many outlying islands.

However, it has now recovered to a populationsize of 400 pairs.

The reproductive fitness of the Mauritius kestrelhas been adversely affected by inbreeding in theearly post-bottleneck population; it has loweredfertility and productivity than comparable falconsand higher adult mortality in captivity.

Not only can bottlenecks in population size bedetected, but also the size of the bottleneckcan often be inferred from loss of genetic diversity.

Northern elephant sealsunderwent a bottleneckdue to hunting with thelast major hunt occurringin 1884.

Subsequently, the population expanded to 350 in1922, to 15,000 in 1960 to well over 100,000 by2000.

During this bottleneck, the seals lost both nuclearand mitochondrial genetic diversity.

Only two mtDNA haplotypes occur in post-bottleneck northern elephant seals, while 23haplotypes are found in related southern elephantseals.

No allozyme variation was found in the northernelephant seals, while the average heterozygosityis 0.03 in southern elephant seals.

The acutal size and duration of the bottleneckis unknown.

Since loss of genetic diversity is related topopulation size, we can estimate the bottlenecksize and duration.

The expected loss of mtDNA diversity is:

t

Ht = H0∏(1 - 1/Nefi) i= 1

Where Nefi is the effective number of females ingeneration i and t is the number of generations from the beginning of the bottleneck until thepopulation is censused.

This equation is similar to one we learned earlierbut 2Ne is replaced by Nef as mtDNA ismaternally inherited and genetic diversity is lostat a rate of 1/Nef.

The mtDNA diversity in southern elephant sealsis 0.980 (assumed to represent H0), while thatin northern elephant seals is 0.409 (Ht).

Many combinations of bottleneck size and durationscan fit these data, but only a limited range willallow realistic growth in population numbers.

A single generation bottleneck would require theeffective number of females to be:

Ht/H0 = (1 - 1/Nef) = 0.409/0.980 = 0.417

yeilding Nef = 1.7

Thus, the effective number of females would beless than 2. This is not compatible with theobserved population Growth, which requires a minimum of about 12 females, unless the ratio ofeffective number of females to actual number offemales is 0.14.

Further, additional genetic diversity is lost duringapproximately 14 generations between the bottleneck and 1960.

Hedrick (1995) assumed that Nef/Nf ratios laybetween 0.25 and 0.125.

The combination of parameters that best fit theloss of mtDNA diversity and the changes inpopulation size were:

(a) A single generation female bottleneck of 12.4 with Nef/Nf = 0.25 (Nef=3.1) or

(b) A bottleneck of three generations with 44 females and Nef/Nf = 0.125 (Nef=5.5).

In both cases, population numbers were projectedTo be close to those observed in 1922 and 1960.

Halley & Hoelzel (1996) reached similar conclusions,Based on detailed computer simulations.

This bottleneck is not sufficient to account for complete absence of allozymic variation.

Therefore, other factors must have come intoplay to explain this loss of genetic variation innorthernelephant seals.

Gene Flow and Population Structure

Genetic management recommendations varysignificantly depending on population structure.

Populations in different habitat fragments may betotally isolated, partially isolated, effectively asingle population, or a metapopulation, depending onthe extent of gene flow and population extinctionrates.

Small and totally isolated populations may experience severe inbreeding.

The delineation of population structure is usuallyonly possible using genetic data.

The degree of population differentiation can bedetermined using FST and related measures forany type of polymorphic genetic marker.

More powerful and informative analyses are possible using gene trees.

Population structure can be identified by mappingsequences of different alleles onto geographiclocations.

The cause of genetic differentiation, restrictedgene flow, past fragmentation, or range expansion can then be determined.

East African populationsof buffalo and impalashow similar FST-valuesof 0.08 and 0.10, respectively.

However, the distribution of mtDNA haplotypes over geographic locations is entirely different in the two species.

12

3

5 21 6 7

17

2

4

9

18

14

2027

13

8

10 16 15

2826 1

19

11

2423

25

22

8 1 6

9

151314

11

16

4 3

5

7

10

12

2 25 17 18 23 19

21 21

20 24

The distribution of Chobe haplotypes (Chobe is most isolated location) is random in buffalobut tightly clustered in impala. Consequently, buffalo exhibit recurrent genetic maternalinterchange between Chobe and more northerly populations.

Conversely, impala have restricted female gene flow that either reflects isolation-by-distanceor isolation of the Chobe population from northern populations.

Sexing of Birds and Mammals

Males and females of many bird species aremorphologically indistinguishable.

Birds must be sexed prior to pairing, as several Cases of “infertile” pairs in zoos have turned out to be two birds of the same sex.

Some mammals are also difficult to sex, especiallycetaceans and secretive species, as it may not bepossible to sex individuals when collecting samplesby skin biopsies, hair, etc. The sex of storedDNA samples may not be known.

Birds have ZZ male and ZW femalesex-determination.

Consequently, PCR primers for W-chromosome specific sequences have been developed to distinguish males from females.

W-specific fragments will amplify from the DNAof a ZW female but not a ZZ male.

Molecular sexing is an important component inthe program to recover the Norfolk Islandboobook owl.

While the program had produced 12 - 13individuals, of which7 were F2, only 2pairs were breeding.

It was unclear whether this was due to hybridsterility, unequal sex-ratio, or individuals of onesex being immature.

As females and males could not be distinguishedby external morphology, a PCR-based techniquewas used to sex the birds.

The population was found to consist of 6 femalesand five males.

A scarcity of mature males was the main factorslowing recovery efforts.

Disease

The disease status of animals is critical inidentifying causes of population decline, and forchecking candidates for translocation or reintroduction.

PCR-based methods provide rapid, reliable andhighly sensitive means for detecting diseaseorganisms.

For example, PCR has been used to study avianmalaria in Hawaii, one of two diseases thought tohave been major factors in the decline of Hawaiianbirds.

Higher-elevation habitats, considered safe frommalaria-carrying mosquitos, have been preservedfor endangered forest birds.

However, Cann et al. (1996) identified malaria inblood of birds from high-elevation habitats onMaui and Hawaii, indicating that these areas are notas safe as previously thought.

Reservoirs of the disease were also detected inintroduced bird species in low-elevation habitats.

Gene trees based upon DNA sequences have beenemployed to determine the source of new diseases.

HIV-1, one of the viruses that causes AIDS inhumans, has been found to be most closely relatedto SIV from chimpanzees, while HIV-2 originatedfrom soot mangabeys.

Similarly, an epidemic causing high mortality inAfrican lions in the Serengeti in 1994 was shownto be due to canine distemper, presumed to have switched species from local dogs.

Recommendations were made to vaccinate local dogsagainst distemper to minimize the risk of repeatepidemics in lions and, especially, in other rarecarnivores.

Diet

Diet is difficult to determine by direct observationin nocturnal and secretive species.

Food items can be identified from faeces by usingPCR with primers specific to suspected food items.

This has been demonstrated in bears, where theplant Photinia was identified as a food item.

The role of predators in causing the decline ofa threatened species has also been assessedusing a PCR-based application.

Microsatellite typing of stomach contents ofglaucous gulls in Alasks revealed that they werepreying on emperor geese, but not on threatenedspectacled eiders (Scribner & Brown 1998).

Since gull numbers have increased, gull predationappears to be the major factor in the decline inemperor geese numbers and their inability torecover.

Gull removal has resulted in improved goslingsurvival.