Molecular Ecology (Modul)two credit lecture

Kristina Sefc & Steven Weiss

Introduction to the Course

Molecular Ecology is a molecular-based approach to answering evolutionary and ecological questions. There is no accepted definition of „Molecular

Ecology“, and it was only a few years ago that a textbook appeared devoted to the subject. There is, however, a peer-reviewed journal Molecular Ecology,

and its description of itself serves as a useful start to understanding what „Molecular Ecologists“ really do.

                         

What is Molecular Ecology?

An Introduction to MolecularEcology

Trevor Beebee&

Graham RoweRC 910 B414

Molecular Ecology publishes papers that utilize molecular genetic techniques to address consequential questions in ecology, evolution, behaviour and conservation. We discourage papers that are primarily descriptive and are

relevant only to the taxon being studied. Studies may employ neutral markers for inference about ecological and evolutionary processes or examine

ecologically important genes and their products directly. Research areas of interest to the journal include:

•population structure and phylogeography •reproductive strategies •relatedness and kin selection •sex allocation •population genetic theory •analytical methods development •conservation genetics •speciation genetics •individual and species identification •microbial biodiversity •genetic marker development •evolutionary dynamics of QTLs •ecological interactions •molecular adaptation and environmental genomics •impact of genetically modified organisms

What is Molecular Ecology?

What are neutral markers?

We can define many of these terms as the course progresses, but thepurpose of this first lecture is to give a background on the ecology of

aquatic organisms (especially fish), and especially those characteristicsthat are most important to understand before being able to conduct fruitful scientific inquiry into evolutionary and ecological processes, using molecular

tools. Our first important lesson here is that regardlessof the technology used, an understanding of the ecology of the organism is a

prerequisite for proper study design and data interpretation.

KNOW YOUR ORGANISM!

Who am I?

We can make a detailed list of what organismal characteristics one should understand before beginning a molecular based study. In truth, any

characteristic may be important depending on the specific question being asked. However, let us first consider what is or is not different about aquatic organisms

in general, and especially those traits that will most likely affect their genetic architecture.

freshwater rivers lakes oceans

The first obvious factor is the environment (habitats) in which aquatic organisms live. We can roughly think of two categories (fresh and salt water) and then

further consider flowing (lotic systems) and still (lentic systems) water habitats of freshwater organisms. Another extremely important characteristic is the thermal

“regime” of the habitat.

About 70% of the earth‘s surface is covered by oceans, and about 97% of all water by volume is contained in oceans. This is compared to 1% surface

coverage for freshwater. Only 0.0093% of the earth‘s water by volume is found in freshwater lakes and streams (the remainder is in the form of ice

and atmospheric gas, etc.). Yet 41% of all modern fish species are found in freshwater lakes and rivers! Why do you think this is so?

Freshwater habitats are highly fragmented systems compared to oceans. Organisms in one lake do not easily reach other lakes. River systems are

constrained in a linear fashion, and aquatic organisms can move along a river channel, but not easily between river channels. This promotes the isolation of

populations.

Reproductive isolation is one of the primary mechanisms promoting speciation. Indeed, the question of whether or not two populations, or phenotypic variants

within an „apparent“ population are reproductively isolated is posed frequently in molecular ecological studies. Reproductive isolation is much

more easily achieved by populations of freshwater organisms, as their habitats are physically isolated from those of other nearby populations. This is the most

basic reason given to explain the high number of freshwater fishes, given the small areal coverage of freshwater compared to saltwater. Still, there are a large number of fish species in the ocean, and populations of organisms can

achieve reproductive isolation in other ways that are not strictly related to the physical separation of habitats. And, freshwater organisms do find ways to

disperse between habitats, or colonize new ones, especially if we consider more historical, glacial, or geological time scales.

Here is a map of a typical catchment area (Einzugsgebiet) of a river

A species can often be found throughout one or more catchment areas, or be limited to only a part of a catchment area. It is always important to ask what is the distribution area of the organism under study, and for freshwater organisms

this is often most easily understood in terms of catchment areas.

This is a figure from a study on the phylogeography of salmonid fishes in eastern Siberia. Note that there are 3 catchment areas outlined, corresponding to three major river systems (Lena, Amur, & Enisey). River basins are physically separated from each other, at least from the perspective of a fish. However, this isolation has a temporal dimension. In this study, a few species were distributed throughout all three catchments, whereas some were limited to one catchment or

the other. How can this occur? How does a species get from one catchment to another?

The structure of basins can change over glacial or geological time scales, and when

they do, opportunities are provided for organisms to disperse from one drainage

system to another. Catchments are dynamic, and every catchment has a

history. But different species have different abilities to disperse. Understanding this

dispersal abilitiy is a key element to understanding an organism‘s evolutionary

history, as well as current demography.

An example of a „river capture“ event (Flußentzapfung)

Such events normally occurover geologic or glacial times scales but can be relevant for

understanding the evolutionary history of many aquatic

organisms. Many such events occurred during the Pleistocene

glaciations, and have largely affected the patterns of

organismal distribution that we see today, at least in temperate

environments.

the upper region of thePugwash river has been„captured“ by the Philip

river

Dispersal abilities of freshwater organismsThe dispersal ability of various fish species, as well as other aquatic organisms

can differ tremendously, and in ways that are not always apparent to the casual observer. One may first consider properties of aquatic environments

themselves, such as the frequency and height of floods, the effect of geological events such as landslides and earthquakes, and the larger-scale dynamic effects of glaciations. But organisms have intrinsic traits and „abilities“ in the form of their mobility and tolerance of dynamic changes. Some fish, for example, are

good at jumping over small waterfalls, or swimming up through swift currents.

coho salmon (Oncorhynchus kisutch)

Other species, such as lampreys, use their sucker-formed mouth and dentition to climb vertical rock walls.

river lamprey (Lampetra planeri)

Bachneunauge

mouth of a seylamprey

(Petromyzon marinus)

When thinking of dispersal ability, it is not only the adult stage of an organism that is important. Many aquatic organisms have egg or other early life history

stages that play a central role in dispersal. Fish have a wide variety of breeding behaviours and reproductive strategies, for example. There are both egg and live-bearing fishes, and among egg layers, some eggs are buried in the gravel of a stream, some drift with currents, and others are adhesive, clinging to substrates of various kinds including plants and woody debris. Such eggs can then be transported by from one waterbody to another, accidentally by birds or other organisms. Some ocean fishes, such as those living on a coral

reefs, may spend their whole adult life in one small area, but the larvae of such species can be carried 100‘s or 1000‘s of kms by ocean currents. In general, dispersal (and thus gene flow) among populations of ocean organisms is very

high, and thus they tend to be more homogeneous than in freshwater environments. Understanding the life cycle of an organism and especially its

dispersal patter, is of fundamental importance to building hypotheses concerning its genetic structure. In fact, dispersal is one of the fundamental

characteristics underlying a species‘s ecolgocial potential. While „real-time“ dispersal is studied using such methods as radio-telemetry or simple mark and recapture, the long term effects of dispersal are understood in genetic terms,

and thus is a task of molecular based research

Round gobyNeogobius melanostomus

An organism‘s dispersal ability can be understood in the framework of current environmental conditions, but the environment is constantly changing. The most rapid changes are usually those instigated by humans. Presently, several species of the genus Neogobius, native to the Black and Caspian seas are

expanding their range up the Danube river, and areappearing in Austrian and Bavarian waters for the first time in recorded history. This expansion is believed to be the result of changes in the shipping industry, that have promoted the use of water as „ballast“. When a cargo ship is empty it must use a large quantity of ballast water to obtain the correct balance and height in the water. When full of cargo, it may need to release ballast water. It is estimated that ballast water can contain up to 3000 species ofaquatic organisms. When this water is discharged, its unknown or unwanted

„passengers“ escape into exotic habitats throughout the world.

The completion of the Danube-Main canal has further promoted the spread of Neogobius, as well as other aquatic organisms throughout Northern Europe, and

even to North America.

The Danube-Main canal, a project first begun by Charlamagne in 793,

was completed in 1992. This created a 3500 km-long international waterway connecting Rotterdam on the North Sea, to the port of

Salina on the Black Sea. Along with the manyeconomic benefits that such a project has provided,the canal has also created a number of environmentalproblems (as predicted) in providing a colonizationcorridor for numerous aquatic organisms.

Zebra musselDreissena polymorpha

Another invasive organism, who‘s spread began long before the constructionof the canal, is the zebra mussel. Zebra mussel is a temperate freshwater bivalve species native to the Black and Caspian seas. The mussels made their way to Western Europe through canals and inland waterways that were used for trade during the Industrial Revolution. Zebra mussels first arrived in the United States around 1985, when transoceanic ships released ballast water into Lake St. Clair of the Great Lakes.

Zebra musselsfouling an outboard

moter

1 meter highaccumulation of

Zebra mussel shells

The European Eel (Anguilla anguilla)

leptcephalus larval stage of a conger eel

Conger oceanicus

10 mm leptocephalus

Both the European and American eel (Anguilla rostrata) spawn in the Sargasso sea, in

100-400 m depths. Their transparent „leptocephalus“ larvae migrate in the Gulf stream for 1-3 years before undergoing metamorphosis and beginning the freshwater stage of the life cycle. This is an example of a catadromous migratory cycle. Anadromous fishes are those that spawn in fresh water, but live as adults in the ocean. Potamodromous fishes migrate within freshwater systems. Both an organism‘s migratory and dispersal patterns have important genetic consequences.

Since all eels spawn in the same place, and their breeding behaviour is that of a „mass spawner“ it was assumed that they constitute a „panmictic population“.

Panmixis is the state where all individuals are mating at random. Panmixia across a whole species is rare in nature. European eels have a very wide

geographic distribution, ranging from the North Atlantic to the Mediterranean Sea, and thus experience a wide range of habitats. Most freshwater organisms with wide geographic distributions consist of numerous described phenotypic variants. But for eels, panmixia was not too difficult an hypothesis to accept. However, this had never been tested with molecular tools, and several recent

molecular based studies have now launched a controversy as to whether eels are really panmictic or not.

Many pelagic ocean fishes are thought to have panmictic or nearly panmictic

populations due to their breeding behaviour, random or chaotic drifting of larvae or eggs,

and more or less physically homogeneous environoment in which they are found.

However, pelagic fish populations can be very large, and difficult to sample, so testing

this hypothesis of panmixia is not easy. A school of herringClupea harengus

World Distribution of Brown Trout

Native range

Introduced

The brown trout (Salmo trutta) has a variable life history pattern, exhibiting both freshwater resident and

anadromous strategies. Brown trout can, however, carry out its entire life cycle in a few meters of an alpine moutain

stream. As such habitats exist throughout its range, and individuals within such populations have little or no

opportunity to migrate to distant habitats, brown trout in Europe are among the most genetically sub-structured

vertebrates that have been studied thus far. For a species, this is the contrasting scenario to panmixia.

The ecology of individuals or populations within their

native range, can differ greatly

from those in introduced habitats.

Life history stages of a pink salmon (Oncoryhnchus

gorbuscha)Alevin

Fry

SmoltOceanphase

Spawningmale

Spawningfemale

The typical salmon has a 5-stage life history cycle. Alevins emerge from the

gravel with their yolk-sac still attached. Fry stage salmon swim freely in small streams, but in the case of pink salmon

they quickly grow into smolts and migrate to sea. After 18 months of rapid growth in the ocean, adult pink salmon return to their natal stream to spawn, completing a strict two-year breeding

cycle. Natal site fidelity (or philopatry) influences genetic relationships among populations, promoting differentiation

and local adaptation. If salmon exhibited 100% philopatry, there would be no dispersal, and no genetic exchange among populations, and this would not

be advantageous for the long-term survival of the species.

The pink salmon‘s life history is very special, even among the seven Pacific salmon species. Its strict two-year cycle results in a interesting phenomena that will help

us introduce an important topic, the population. A population is a very important concept in evolutionary biology, as it is the unit that evolves. But, what is a

population? If a species of fish lives in a small lake, and all the individuals in that lake had an equal probability of mating with each other (panmixia), than the lake would serve as a perfect physical unit describing the population. Another nearby lake would constitute another population, with no possibility of confusion if, 1) the lakes were not connected by some stream or river (nearby lakes often are), 2) birds

or other animals do not carry fish or their eggs from one lake to the other (they sometimes do), and 3) humans are not transporting fish from one lake to another (given the chance, they will always do this). The same concept can be more or less

suitable in rivers and streams, except it can be much more difficult to define physical borders as stream systems belong to complex of linear networks that are all connected to each other (catchment concepts). You should begin to see that the

„physical“ dimension of a population is not the only factor, there is a temporal dimension that is very important. Given time, individuals of one population will almost always manage to exchange genes with individuals of another population. Lets return to the pink salmon. In every stream where there are pink salmon, no matter how small and isolated, there are always two very distinct populations of

them. Why?

One more important factor in the pink salmon story is that pacific salmon have a life history strategy that is semelparous. Semelparity means that an organism dies after its first reproduction, where as iteroparous organisms mate repeatedly over their life-span. If an organism is semelparous, and confined to a strict two year

cycle, there is an interesting temporal affect on its genetic structure. A pink salmon born in 2002 will spawn in 2004, and its genes will be passed on to

generations of salmon in 2004, 2006, 2008, etc. But what about the odd years? Are there no salmon in 2003, 2005, etc.? Some stream populations of pink salmon

have only adults returning to spawn in odd or even years, but many have both. These odd and even year fish constitute separate populations, because they are reproductively isolated, even though they live in the same place, and would not have any „biological“ barrier to interbreeding. Remember again the European eel. „populations“ (in the physical sense) of eels from the North Sea are a long

way from those in Greece, and these environments are also completely different. But, if all breeding activity occurs in one place, and if random mating is the rule, then the entire „species“ may consitute one genetic population. The lesson here is

that populations appear to be quite different from species to species. One must understand the life-cycle of the organism, its breeding behaviour, its range, and its pattern of dispersal before trying to define a population. Second, populations are

not discrete inflexible units, but are rather dynamic across both spatial and temporal scales.

Populations, cohorts, & individuals

In the example of salmon (semelparous), all individuals of a given cohort (year-class) can be followed as a unit through their entire life-cylce, and thus consist of

a population or genetic „unit“ that is easily defined. There are many benthicinvertebrates, such as stoneflies (Plecoptera), caddisflies (Trichoptera), and

mayflies (Ephemeroptera), that are semelparous, and have discrete life-history stages, and so each indvidual can easily be assessed in terms of its „year-class“.

Summer stone flyClaassenia sabulosa Western March Brown

Rhithrogena morrisoni

American GrannomBrachycentrus americanusGiant red sedge

Pycnopsyche scabripennis

Each year class, or life-history stage in such organisms can be consideredas a discrete population unit in genetic terms and such a distinction can become

useful depending on the question being asked (at the very least, samples should be taken from different stages, to insure that population genetic

estimates are not biased). However, in typical iteroparous organism there isoverlapping generations and sometimes a very complex and/or cryptic age

structure to the population. This is particularly true with long-lived freshwater fishes. The use of molecular genetic tools to assess the population structure of

marine fish populations, for example, is made more difficult by this complex age-structure, among other cryptic variables. Later in the course, you will here the

term „effective population size“ along with other population genetic principles (e.g Hardy-Weinberg Equilibrium), most of which have been developed and explored

in model or theoretical systems with a simple structure (such as a randomly mating, non-overlapping generations). The real world is more complicated,

however, and these complications limit and challenge the tasks of a Molecular Ecologist. The purpose of this introductory lecture is simply to introduce the

„organism“ and its complexity, and emphasize that a researcher should not loose site of the organism, while exploring its genome.

Things to ask about your study organismWho am I?

European graylingThymallus thymallus

What is my distribution? Throughout cool water reaches of European rivers from northernmost Scandinavia, east to the Ural moutains in Russia, south to

former Yugoslavia, including Adriatic drainages of Slovenia and northern Italy, and west to the upper Loire River basin in southwest France

Repeat spring spawner, primarily in lotic habitats (iteroparous)Short-distance migrations (to 50 km?), but a poor jumper

Lives in both rivers and lakesgenerally salt-intolerant (some populations in the Baltic Sea)

high-population structuringauto-tetraploid ancestory (see ploidy?)

What is my ploidy?Who are my relatives?

What is my life

-cycle like?

Am I hard to:

Find?

Catch?

Raise at home?

What is my reproductive

strategy?