Ex Situ Plant Conservation

Certificate Program in

Native Plant StudiesDecember 12, 2009

Johnny Randall

North Carolina Botanical GardenUniversity of North Carolina at Chapel Hill

Topics covered:

Ex situ, definition, history, and global context

Prioritization

Components of an ex situ capability

Seed collection

Seed storage

Reintroduction

Ecotypes and seed zones

Some further examples

Definitions

Situ is from Latin and refers to place

In situ means „in the place‟

Ex situ means from, or away from the place,

Saving seeds off site, out of nature, as a means of conserving the sampled populations and taxa in the wild can seem like a contradiction in terms (but, properly used, it is not)

From BGCI web page…

Ex Situ Conservation

Ex situ conservation is the conservation

and maintenance of samples of living

organisms outside their natural habitat,

in the form of whole plants, seed, pollen,

vegetative propagules, tissue or cell

cultures.

Evolution of ex situ conservation

• Botanic garden as repository for academic study – 1400s to 1900s

• Botanic gardens first mentioned as “Jardin Conservatoire,” supporting wild plant diversity by Cugnac in 1953

• Botanic gardens as “Arks” – 1970s

• Botanic gardens as partners in “Integrated Plant Conservation” –1980s to present

Off site, or Ex situ

Conservation resources are...

1. A means to an end (enhanced

survival prospects in nature, and)

2. A part of a whole (Integrated

conservation strategies, in which

ex situ methods support in situ

efforts)

Seed Bank History

Vavilov first to recognize value of genetic diversity,

created first modern seed bank in St Petersburg, Russia

USDA NCGRP – crop and other seed bank back up

Doomsday Seed Bank

Millennium Seed Bank – RBG Kew

Seeds Of Success/BLM – US native species

Center for Plant Conservation - for rare plant species

Kuijt I, Finlayson B PNAS 2009;106:10966-10970

©2009 by National Academy of Sciences

Oldest known granaries 11,000 years ago in Jordan

River Valley, predating domestication by 1,000

years or so, and together set scene for sedentary

life style and dramatically increased local

population sizes.

Structure 4, phase 1, Dhra′, Jordan looking north

©2009 by National Academy of Sciences

Success is what me make of the

mess we‟ve made of things.

T.S. Elliot

The mess being the current state of the global

environment: massive loss of habitat and biodiversity,

degradation and fragmentation of habitat, rapidly

increasing rates of extinction, invasives, etc. All of

which are a consequence of the huge human

population – which itself was made possible by

agriculture, which in turn was made possible by seed

storage. Thus, seed banks contributed to what got us

into this mess...

Scientists in Kabul reported the loss of Afghanistan's

principal agricultural insurance policy: two stores of

carefully collected seeds, materials selected to

represent the genetic diversity of native crops.

It was a looting of the worst kind - a theft of that

agrarian country's stockpiled agricultural heritage. In it

were seeds to help that nation's 22 million people

rebuild the capacity to feed themselves.

Ironically, the stores were not plundered for those

plant materials; the seeds were dumped in disarray

onto the floor of ransacked buildings in two cities. The

looters merely ran off with the airtight plastic and

glass jars in which the seeds had been kept.

N.I. Vavilov• First to recognize value of genetic diversity.

• Established first modern seed bank in St Petersurg (now

Leningrad)

Centers of origin for food plants

The USDA seed storage effort, used to be called

National Seed Storage Laboratory. Historically, their

main focus has been on agriculturally important plants

and their wild relatives. Leading laboratory for seed

storage science.

Millennium

Seed Bank

Project

15 Oct 2009

Kew‟s Millennium Seed Bank

partnership is celebrating

collecting, banking and conserving

10% of the world‟s wild plant

species by banking its 24,200th

plant species.

Seeds of Success

BLM originally in conjunction with

Millennium Seed Bank.

Attempting to gather

large samples of more

commons species.

Millennium Seed Bank /

Seeds of Success Project

Wiregrass collection in NC

Coastal Plain with TNC

and NC Plant Conservation

Program partners

Seed

Processing

Sending

seeds and

vouchers

to Kew…

The CPC Participating Institutions

Components of an Ex situ (off site)

Conservation Capability

I. Collect genetically representative seed samples

II Keep seeds alive for a long time, and learn to germinate and grow them

III. Use samples as population founders when needed

19911996

2004

1. Collect genetically representative samples.

2. Maintain samples off site in good

condition for a long time, and be

able to use them if needed.

3. Use samples to

reintroduce genetic

material to the wild if

necessary.

Center for Plant Conservation

Berry Botanic Garden Seed VaultVented exterior door with exhaust fan

Freezer

Fluorescent lights

Fluorescent lights

Wire shelving

Ceiling mounted low profile

condensing unit for temperature

control

Fire doorInsulated door

Vestibule Entry

Scale 5 feet (ca 1.5 m)

Steel reinforced Concrete shell

Insulated shell

Compressors:

Vault

Freezer *

**

* Thermostat for Power Cut-Off Safety

** Hygrostat

Dehumidifier

Vault temperature controller

Biodiversity Concentration in US

• Total U.S. plant species ~20,000

• Extinct >200

• Fed. listed endangered, 889

threatened, or qualify (~5%)

• Species of concern (25%) > 5,000

Mountain Sweet

Pitcher Plant

(Sarracenia jonesii)

Vulnerability in the U.S. Flora

Condition of Listed Plant Species

(survey of recovery plans 1982-1998)

Solidago plumosa

One Site Remaining

•65% of listed species had < 10 sites remaining;

49% had < 5 sites

•74% had less than 100 individuals per site. These

populations are VERY vulnerable to chance events

and genetic erosion—they could be extinct within 20

years or less without intervention

Botanic gardens and arboreta:

• Develop and manage a documented

collection of plants…they represent

some of the most concentrated sites

of species richness

• Are evolving from “collections of

curiosities” to centers of research and

conservation

• Serve as “windows to the plant world”

• Collectively host over 150 million

visitors per year…serving as centers

for outreach on plant conservation is

an area where we can make a unique

and significant impact

The ex situ/in situ continuum• Cryopreservation

• Seed Banking

• Tissue Culture Storage

• Tissue Culture Propagation

• Cultivation in dedicated conservation facility

• Specialist cultivation in controlled environment

• Cultivation in mixed display or reference collections

• Field gene bank

• Commercial Cultivation

• Community Garden

• Inter situ

• In situ - horticulturally managed individuals

• In situ - managed wild populations

What is ex situ delivering?

• 1800 botanic gardens hold 2.5 million accessions, representing 18,000 taxa

• About 100 taxa exist only in botanic gardens

• Many ex situ collections vastly outnumber surviving wild populations

• In the US, over 80 rare taxa are being reintroduced

Why isn‟t ex situ delivering

more?

• A perception that it undermines

in situ conservation efforts

• A lack of professional confidence

that gardens can hold diverse

samples for long periods of time

• Conservation “liabilities” - source

of pathogens, genetic change,

etc.

Characteristics of many botanical

garden collections

• Small, often from a few closely related founders

• Subject to fluctuating population size

• Little or no ecological/biological information

• Little information on the history of the taxa in cultivation

• Individuals susceptible to artificial selection, genetic drift, inbreeding and hybridization with congeners

• Persistence in collections highest for horticulturally amenable taxa

Ex Situ Target from the Global

Strategy for Plant Conservation

• TARGET 8: 60 per cent

of threatened plant

species in accessible ex

situ collections, preferably

in the country of origin,

and 10 per cent of them

included in recovery and

restoration programs.

CPC National Collection at

The North Carolina Botanical Garden• Amaranthus pumilus

• Arabis serotina

• Asplenium heteroresiliens

• Buckleya distichophylla

• Carex lutea

• Clematis socialis

• Clematis viticaulis

• Coreopsis latifolia

• Echinacea laevigata

• Eupatorium resinosum

• Geum geniculatum

• Helianthus schweinitzii

• Iliamna corei

• Isoetes tegetiformans

• Kalmia cuneata

• Leiophyllum buxifolium

• Liatris helleri

• Lilium grayi

• Lobelia boykinii

• Lysimachia asperulifolia

• Oxypolis canbyi

• Parnassia caroliniana

• Paxistima canbyi

• Pityopsis ruthii

• Ptilimnium nodosum

• Rhexia aristosa

• Rhus michauxii

• Sagittaria fasciculata

• Sarracenia jonesii

• Sarracenia oreophila

• Solidago spithamaea

• Solidago verna

• Solidago villosicarpa

• Stylisma pickeringii var. pickeringii

• Thalictrum cooleyi

• Vaccinium sempervirens

Carex lutea – Golden Sedge

Solidago villosicarpa

Recent additions

to CPC National

Collection taxa

in need of study

Clematis viticaulis

Buckleya distichophylla

Paxistima canbyi

Prunus alleghaniensis

Leiophyllum buxifolium

Collections transferred

from The Arnold Arboretum

of Harvard University

Seed collection and

storage projects

CPC and NPS Collections•Geum radiatum

•Liatris helleri

•Aster georgianus (Symphyotrichum georgianum)

•Amaranthus pumilus

•Sarracenia oreophila

•Ptilimnium nodosum

• Lilium pyrophilum

Alex Reynolds

USFWS Flex Grant•Liatris helleri

•Hudsonia montana

•Parnassia caroliniana

•Amaranthus pumilus

•Delphinium exaultatum

•Asplenium heteroresiliens

•25 collections scheduled for 2008 in 8 states.

Mean Seed Mass Variation

in Amaranthus pumilus

•Throughout geographic range

•18% from NJ-SC

•Among populations within

states

•21% within NJ

•31% within NC

•Among plants within a

population

•21% among plants at

Indian Beach, NC

Ex situ capacity vs. need -

hotspots

Ex situ load for selected

countries

Country Plant

endemics

(mean)

60%

stored

10%

managed

# BGs Required

BGs (@25

taxa /BG

for 10%)

Brazil 17,500 10,500 1750 29 70

Indonesia 16,650 9990 1665 5 67

Madagascar 9200 5520 920 2 37

China 10,000 6000 1000 106 40

USA 4000 2400 400 296 16

Practical recommendations

• Improve professional

capacity

• Refine species selection

• Support in situ management

• Improve fund raising and

outreach

• Manage ex situ “liabilities”

The challenges of prioritization!

Prioritization – Broad Scale

• The five “E‟s”

– Endangerment

– Endemism

– Ecological

– Economic

– Emblematic/Education

Prioritization – fine scale

• New England model

(Farnsworth, et al., Biological

Conservation, 2006)

• A set of 3 decision matrices

that rank species and

populations for ex situ

activity

Ex situ usually not 100% efficient.

Original

PopulationEx situ

New,

Reintroduced

Population

Gen

etic D

ivers

ity

Time?

Genetic Loss

?What are sources and magnitude of losses?

Matrix 1

• Are seeds/spores orthodox?

• Is vegetative propagation possible?

• Can seeds be germinated and grown?

• If no, is collaboration with research facility

arranged?

• Are there 5 or fewer populations?

• Is current reproduction sufficient?

Matrix 2

• G-rank

• Flora Conservanda rank

• Range outside of NE

• Total EO‟s in NE

• In NEPCoP seedbank?

• # EO‟s in seedbank?

• Proportion of accessions confirmed viable

Matrix 3

• Reproduction consistent

• Population outlier or middle of range

• Protection status

• Habitat typical?

• Imminent threat

• Landowner permission expected

• Collection possible given current

reproduction

Developing species

conservation plans• Are ex situ activities

appropriate and needed?

• If so, what techniques?

Seed banking as an “insurance policy” for orthodox taxa?

Alternative techniques for recalcitrant taxa?

Reintroduction/translocation?

Clues that ex situ activities are

needed:

• Small populations (size and/or number decreasing)

• Increased fragmentation/isolation

• Variable environment or current stress (drought)

• Threats imminent

• Habitat highly degraded

• Genetic or demographic problems

• High priority species

Plants aren‟t reproducing…why?

• No seeds….

– Lack of pollinators

– Lack of compatible mates

• No seedlings…

– Poor conditions for

establishment

– Fruit dispersal agent important

– Severe inbreeding

Talking it out

Sampling for diversity

• Capturing genetic

diversity is important for

conservation programs

• Genetic diversity allows

plants to avoid inbreeding

and is a buffer against:

changing environment

pathogen attack

demographic stochasticity

How many populations should be

sampled?

• Usually 1-5, more if:

High diversity,

limited gene flow

between populations

Destruction of

population imminent

Recent or

anthropogenic rarity

How many individuals should be

sampled?

• Usually 10-50, more if

high genetic diversity

among individuals

within populations

• Sampling follows law

of diminishing returns

How many seeds/individual should

be sampled?

• Usually 1-20, more

if:

high attrition rates

planned

reintroduction

Multi-year collections are indicated

if:

• Low annual

reproductive output

• High year to year

variation in population

size or structure

• Multi-year collections

may capture more

genetic variation

First do no harm

• Sample=trample

• Permits and landowner

permission must be in

place

• Site confidentiality

• Unintentional introduction

of weeds/diseases

• Sample size

considerations

Avoiding harm to native

populations

• Menges, Guerrant and Hamze (2004) modeled demographic consequences of 36 patterns of seed harvest:– 10, 50, and 100% of fecundity for

10, 50, and 90% of years on populations of 10, 50, 100, and 500 plants

– used published projection matrices from a range of taxa with different life histories

Avoiding harm - conclusions

• Menges, Guerrant and Hamze suggest 3 rules:– harvesting 10% of seeds in 10% of

years (or less) is generally safe

– harvesting 50% of seeds in 50% of years (or more) is generally unsafe

– less intense, frequent harvests are safer than more intense, infrequent harvests

• Long-lived woody taxa and large populations are less sensitive to seed harvest

Seed collection conclusions

• Assess extent of population and estimate fecundity, 5-10% of fecundity should be maximum collection amount

• Collect 1000+ seeds (20 seed from each of 50 plants) if population can withstand it (if it makes more than 20,000 seed)

• Sample in a stratified random fashion

• High attrition rates are the rule, rather than the exception

• Consider a multi-year collection strategy

• Adjust as needed depending on purpose of collection (Reintroduction? Long-term storage? Research?)

Seed curation for orthodox

seeds Collect into paper or cloth bags, keep cool and

dry

Clean as soon as possible

Keep maternal lines separate

Dry seeds – drying temperature influences

appropriate RH for desiccation chamber

• At 25C, desiccate at RH = ~38%

• At 15C, desiccate at RH = ~46%

• At 5C, desiccate at RH = ~32%

Store in airtight containers at –18C

Test viability immediately & at 10 yr. intervals

Relationship between drying

temperature and humidity

• Drying relative humidity and temperature combinations to achieve 20% relative humidity at storage temperatures (Walters, 2004)

Dealing with recalcitrance

• Cryopreserve large

numbers of individuals if

possible

• Alternatively, consider

slow growth tissue

culture, inter situ

techniques, field gene

banks

Redbay Ambrosia Beetle,

Xyleborus glabratus

Eichhoff (Scolytinae:

Curculionidae)

Laurel Wilt Disease

Butternut Canker(Sirococcus clavigignenti-juglandacearum)

Hemlock

wooly

adelgid

Tissue culture projects with Cincinnati

Zoo and Botanical Garden

-Sagittaria fasciculata

-Isoetes tegetiformans

-Lobelia boykinii

-Parnassia caroliniana

-Asplenium heterosiliens

Maintaining genetic integrity

• The genetic structure

of a group of plants in

a garden will change!

• Minimizing genetic

change is crucial for

conservation

programs.

Three major genetic risks in ex situ

collections

• Genetic Drift

• Adaptation to

cultivation

• Mutation

accumulation

Genetic drift

• Random loss of alleles in

small populations

• Homozygosity increases,

some alleles become fixed

• Worsens as Ne decreases

How do we counter effects of

drift?

• Increase effective population size (Ne)

– Equalize family size

– Equalize sex-ratios

– Equalize numbers in different generations

• Provide immigration from wild

populations

Evidence for local adaptation

• Fertilizer dependence in Anthoxanthum

• Herbicide resistance in many taxa

• Moisture/temperature morphotypes in Avena

barbata

• Sun/shade differences in Plantago lanceolata

• Competition-induced differences in Veronica

peregrina

• “Loss of drought tolerance in Solidago

plumosa”…

When can unconscious

selection occur?

• Seed collection

• Seed storage

• Germination

• Propagation

• Growth in nursery or

greenhouse

How can we minimize effects of

artificial selection?

• Equalize family size (relaxes selection)

• Minimize time in captivity

• Immigration from wild populations

Mutation accumulation

• Relaxing selection can allow

deleterious mutations to

accumulate

• Mutations accumulate

independent of pop. size, but

fixation by drift depends on Ne

• Fitness declines depend on

shape of fitness function

Minimizing genetic change

• Equalize founder representation

• Minimize generations in captivity; avoid grow outs when possible

• Maintain large population size

• Provide periodic immigration from wild

Genetic guidelines for

reintroductions• Create large, genetically-diverse

populations

• Equalize founder representation

• Use stock from the same

ecoregion and/or evolutionary

lineage

• Consider the taxon‟s breeding

system and historic patterns of

gene flow

• Design restoration as an

experiment

Multiple vs. single source stock

• Introductions

– Use multiple sources

• Reintroductions

– Use propagules from that site unless

they lack diversity

• Augmentations

– Use only propagules from that site

unless circumstances are

extraordinary

Ecological Connections

• Pollinators

Are they present?

Is population large enough to

attract sufficient numbers?

• Seed dispersal agents

• Mycorrhizae

• Others (rhizobia, host plants,

nurse plants)

Other factors to consider

• Abiotic ecological

considerations

Soils

Disturbance regime

Hydrology

• Political considerations

Land ownership

Land management

Outplanting

• Consider benefits of hard

vs. soft release

• Develop an experimental

design

• Monitor, Monitor, Monitor!

• Share results and

celebrate successes!