Biol 119 – Herpetology Lab 4: Phylogeny Exercise, Anuran Diversity Fall 2013

Philip J. Bergmann

Lab objectives The objectives of today’s lab are to: 1. Continue to familiarize yourselves with local herps and their external anatomy. 2. Use this knowledge to collect morphological character data from specimens. 3. Reconstruct a phylogeny using your collected character data. 4. Be able to discuss phylogenetic concepts introduced in lecture in the context of the lab. 5. Familiarize yourselves with extant diversity of the Anura. Collecting data from specimens and using it to reconstruct a phylogeny will reinforce your knowledge of local herps and accustom you to using the terminology from last week’s lab. During today’s lab you will also begin studying amphibian diversity. The lab will introduce the Anura (frogs).

Tips for learning the material Take some time today to review material that was covered last week. The same specimens are out to allow you to do this. Use the opportunity to quiz yourself or a partner by covering up species names and identifying specimens. Note which ones you find easy to ID and which ones give you trouble. Why do those give you troubles? Refine your criteria for differentiating them and take some more notes.

However, do not spend too much time reviewing last week’s material – there are new things to cover as well. Today’s phylogeny exercise will help you to reinforce some of this material, at least for the frogs, which will be the focus of that exercise. Although the Anura has a conserved body plan – all are rather short and rigid bodied, with well-developed limbs, there is an incredible amount of diversity. Pay close attention to some of the special external anatomical traits that characterize the groups of frogs you see today. Remember the strategies you have been using to learn the species that are available in lab.

Starting with this lab, species names that are in bold represent Massachusetts natives, which you will have to know how to identify to species. However, the species that are not in bold are not local and you are only responsible for knowing which higher taxon/clade (“family”) they belong to.

1 Biol 119 – Lab 4: Phylogeny & Anura Exercise 1: Collecting character data A necessary step toward constructing a phylogeny is to collect character data. This means looking at specimens and figuring out which characteristics they posses. In practice, this is rarely straightforward. First, a researcher must look at many (possibly hundreds) of specimens and figure out which ones are variable enough but not too variable to be useful. Characters that don’t vary among any of the species or operational taxonomic units (OTUs) are plesiomorphic and not informative of phylogeny. Likewise characters that characterize only one OTU are called autapomorphic and are also not useful to reconstructing phylogeny. Autapomorphies, however, are useful for identification or diagnosis of species. Throughout the labs, by learning how to identify species that you see, you will frequently rely on autapomorphies – what makes each species unique. At the opposite extreme are characters that vary too much. These are often polymorphic within species. Although they sometimes can be useful for phylogenetic reconstruction, they are typically too variable. For phylogenetic reconstruction, synapomorphies are the characters of interest. Synapomorphies unite some but not all of the OTUs.

Take a look at the turtles that are on display. What is a synapomorphy of the clade Emydidae (three local turtle species belong to it)?

What is a plesiomorphy of the Emydidae? (This is a trick question!)

Now take a look at the snakes on display. What is a plesiomorphy of Thamnophis ?

What is a synapomorphy of Thamnophis ?

What is an autapomorphy of Thamnophis sirtalis ?

Repeat this exercise with other local species on display so that you gain an understanding of what these terms mean and how they can help you learn features of the taxa you are learning.

In today’s lab, you DO NOT need to spend time selecting characters. This is very time consuming and has been done for you. You can concentrate on examining a small number of specimens of each species and coding your findings into the table provided below. We will be working on a phylogeny of some local frogs to help you learn to differentiate them.

Characters generally come in two types: binary and multi-state . Binary characters only have two states (written in a matrix as 0 when ancestral and 1 when derived ). All the characters we are using today are binary. Multi-state characters have more than two states. For example, if the

2 Biol 119 – Lab 4: Phylogeny & Anura character is number of scale rows, the states might be 48, 49, 50, or 51 (in this example there are four states).

For the turtles on display, come up with one example of each type of character listed: Binary:

Multi-state:

What is meant by "ancestral" and "derived"?

Complete the table provided using the local frog and salamander species on display. Some of specimens have capital letters beside them (A-I). Fill in the species names in the appropriate row, then code all of the characters (1-10). Character descriptions are supplied below the table, as well as instructions on how to code each one. Pay attention, some characters are very subtle!

Character data worksheet Character ID Species name 1 2 3 4 5 6 7 8 9 10 A B C D E 1 F G H I

Character descriptions 1. If the species has a parotid gland, then code it as "1"; if it lacks one, code it as "0".

2. Toes on hind limbs are obviously webbed (1); or free from each other (0).

3. Adhesive pads on distal tips of toes present (1); or absent (0).

4. Brownish blotches on body and hind limbs numerous and regular (1); or not (0).

5. Dorsolateral folds are present but are partial or incomplete (1); or they run continuously from posterior to the eye to the groin, or are absent (0).

3 Biol 119 – Lab 4: Phylogeny & Anura 6. Four limbs present, with at least three digits on each (0); or fewer digits (1).

7. Tympanum prominent but not fully encompased in a brown postocular blotch (1); or otherwise (0).

8. External tail present (0); or absent (1).

9. Brownish blotches on body and hind limbs are rectangular in shape, forming a reticulated pattern (1); or blotches are ovular, diffuse, irregular, or absent (0).

10. Skin warty (1); or relatively smooth (0).

For which species was it hardest to code the characters? Why?

For which species did you find it easiest to code characters? Why?

Exercise 2: Building a phylogeny Once we have a character matrix, we can go about producing a phylogeny. Before we do that, however, look at your character matrix to see how the characters are distributed. It should be obvious that different characters have different distributions across taxa. Think about them in the context of the types of characters introduced earlier and in lecture.

List the numbers of the characters that are plesiomorphies.

List the numbers of the characters that are synapomorphies.

List the numbers of the characters that are autapomorphies.

How many of the 10 characters are useful for reconstructing phylogeny using a parsimony approach? Which ones? Why?

4 Biol 119 – Lab 4: Phylogeny & Anura Quite often characters are coded in such a way that zeros (0) are interpreted as ancestral and other character states are derived. The most popular way of determining polarity of characters (which states are ancestral vs. derived) is through the use of an outgroup.

From looking at your character matrix, which species is the outgroup? Explain.

Now that we have identified which characters are useful in reconstructing the phylogeny and which taxon is the outgroup, we can go about making a phylogeny. As mentioned earlier, synapomorphies are used in reconstructing phylogeny because they unite some but not all taxa. These are the characters we will be using today.

There are several ways to build phylogenies. Today we will use the algorithmic approach, which works well for a small matrix like the one you created. Go through the steps below to first make a venn diagram , and then to draw a phylogeny from your matrix (use the space provided on the next page and a pencil so that you can erase things if needed:

1. Select all characters that unite only two OTUs and list these taxon pairs.

2. Are any OTUs represented in >1 pair? a. If yes, list all of the OTUs together. b. If no, go to #3

3. In the space provided, write the names of OTUs belonging to each pair/group together and draw a circle around each pair/group.

4. For each pair (or group of OTUs), search for a character that unites that pair with another single OTU or pair/group that you have already defined. a. If a single OTU is added, write its name just outside the circle of the pair/group it is allied with and draw another circle around the previous circle and the new taxon/taxa. b. If two of your already-defined pairs/groups are united by a character, then draw a larger circle that goes around both groups’ circles.

5. Repeat steps 2-4, working from the least inclusive characters (those with fewest 1’s) to most inclusive characters (those with most 1’s) until you run out of synapomorphic characters.

5 Biol 119 – Lab 4: Phylogeny & Anura Use the space below to work on your venn diagram.

Congratulations, you have now made a venn diagram, which is another representation of a phylogeny. Each circle coincides with a node and associated clade on the phylogeny.

Use the space below to draw your phylogeny, using your venn diagram for guidance.

6 Biol 119 – Lab 4: Phylogeny & Anura Now that you have a phylogeny, take some time to consider whether it makes intuitive sense. Are the species related to one another as you expected? As you will learn throughout the semester, phylogenies are not only useful, but necessary to study the evolution of any sort of trait you might be interested in. For now, consider your phylogeny.

Is the phylogeny fully resolved?

What is a ‘polytomy’?

What approach would you take to resolving the polytomy?

Using your phylogeny and character matrix, which relationship is most strongly supported? Why?

How might the autapomorphies that you coded be useful?

Why couldn't we use the autapomorphies to help make the phylogeny?

Map each of the ten characters onto your phylogeny. You can do this by drawing a line through each branch of the phylogeny where a character changes states and labeling the line with the character number. This allows you to better see how the characters have changed through evolution.

7 Biol 119 – Lab 4: Phylogeny & Anura Exercise 3: Anura diversity General Information Frogs are a monophyletic group comprising the clade Anura. Salientia includes both extant and extinct frogs. Frogs have been around since the Triassic (~230 ma). Currently, we recognize over 300 extant genera and ~4,800 species that are placed into 29 major clades (See Fig 3-20 from Pough et al. 2004, left), making frogs the largest group of amphibians. Several clades of frogs are found on all continents except Antarctica. They reach their greatest diversity in tropical areas, particularly in South America and Africa. Eight major clades are found in North America, four of which are native to Massachusetts. More than half of the world’s frogs belong to the "Hylidae", "Leptodactylidae", and "Ranidae", all of which are probably not monophyletic. Although relationships among some of the more basal clades of frogs are well resolved, those of more derived groups are ambiguous (see to left).

Which of the major clades of frogs on the phylogeny to the left have members that are found in Massachusetts?

Generalized morphology Frogs have four limbs, a short body, a large head, an internalized tail, very short ribs, 9 or fewer presacral vertebrae, hind limbs larger than front limbs, a fused radius and ulna, a fused tibia and fibula, elongate ankle bones, and the ability to project their tongue. Many of these traits are specializations for saltatory locomotion.

Generalized life history The eggs of all amphibians lack a shell and extraembryonic membranes that are characteristic of amniotes. As a result, amphibian eggs are not resistant to water loss and are laid either directly in water or closely tied to water. Most amphibians also lack internal fertilization. Anuran mating typically starts with males calling from a selected location announcing their presence to receptive females. Generally males congregate at a source of water during the spring and call.

8 Biol 119 – Lab 4: Phylogeny & Anura These aggregations are known as choruses . A female approaches a selected male and the male mounts the female’s back and grasps her in a mating hold known as amplexus . The pair enters the water and either the female lays eggs in the water at the same time as the male deposits his sperm over the egg mass, fertilizing the eggs; or the female takes up a quantity of water for her to include in the egg jelly for deposition at a terrestrial location overhanging water. As she lays the egg mass over the water the male deposits his sperm over the egg mass. Some species practice a modification of the chorus called explosive mating aggregation , where both males and females converge in large numbers in a small area for a short period of time.

Eggs develop into larvae called tadpoles and either hatch directly in the water or drop into the water from the overhanging egg mass. Tadpoles are typically aquatic and eat algae. Larvae in anurans are very different ecologically, behaviorally, and morphologically from adults and are often under entirely different selective pressures. After about two weeks to well over a year, depending on the species, the tadpoles undergo major changes as they metamorphose into frogs. They develop limbs, lungs, bones, teeth, true jaws, a tongue, and a digestive system designed for carnivory. They also absorb the tail. Some species are direct-developing and skip the larval stage.

Under what conditions would you expect explosive mating aggregations to be common?

Some notes on anuran identification The following characteristics can be used to identify different species by determining if they are present or not, and by noting their attributes if present.

Toads and Spadefoot Toads: • Size and shape of parotoid glands • Size and shape of cranial crests • Size and shape of tubercles • Size and shape of spots/botches • Number, “color”, and location of warts Frogs: • Length and “location” of dorsolateral fold • Size and location of spots • Eye and mouth stripes • Toe pads • Relative size of tympanum/ear drum • Tubercles in skin • “Coloring” under hind limbs

9 Biol 119 – Lab 4: Phylogeny & Anura North American Clades Bufonidae: True Toads Content and distribution: 33 genera, 455 species. Almost everywhere except extreme northern latitudes, NW Africa, and Australia (where one species is introduced). Two species are native to Massachusetts.

Morphology: Most have numerous wart-like glands that secrete sticky white poison, which can paralyze or kill other species. The most conspicuous of these glands are the parotoid glands, which are useful for identifying species. All bufonids lack teeth. Many have cranial crests which are also useful for species identification. Color may change from light to dark in response to temperature.

Life history: Most are fairly terrestrial, but return to ponds and streams to mate. Most bufonids are oviparous, but a few are ovoviviparous.

Miscellaneous facts: Bufo marinus , the cane toad, has been widely introduced around the world and has wreaked much ecological havoc, especially in Australia. One of the species on display, Incilius alvarius , has parotoid glands that contain one of the most powerful hallucinogens in the natural world. Toads demonstrate both explosive and chorus breeding behavior.

Species in lab: Anaxyrus americanus - American Toad Anaxyrus cognatus - Great Plains Toad Anaxyrus fowleri - Fowler's Toad Anaxyrus sp. - An unidentified species Incilius alvarius – Sonoran Desert Toad

For the two bufonid species listed above in BOLD, list one autapomorphy that you can use to distinguish it from the other species. Use the space above.

"Hylidae": Treefrogs Content and distribution: 40 genera, 835 species. North, Central, and South America, Europe, north Africa, Middle East, China, and Australasia. Two species are native to Massachusetts.

Morphology: Most hylids have well-developed toe disks that work through wet adhesion to allow them to climb smooth vertical surfaces. Some have skulls modified for burrowing. They have slim waists and long legs.

Life history: As the common name suggests, most hylids are arboreal. However, most return to water to mate. Some species are burrowers. Look at the toes and head shape to determine whether a hylid is a climber or burrower. Hylids are generally chorus breeders.

10 Biol 119 – Lab 4: Phylogeny & Anura Species in lab: Acris crepitans – Northern Cricket Frog Hyla arenicolor – Canyon Treefrog Hyla cinerea – Green Treefrog Pseudacris crucifer – Spring Peeper

What is one autapomorphy of Pseudacris crucifer?

What other hylids live in Massachusetts?

Microhylidae: Narrow-mouthed Toads Content and distribution: 69 genera, 350 species. Southern U.S., Central and South America, sub-Saharan Africa, SE Asia, northern Australia.

Morphology: Most are relatively small. New World species generally have very narrow mouths, pointed heads, teardrop-shaped bodies, and little or no webbing between their digits. They have a fold of skin across the back of the head, and a distinct pattern of palatal folds in the mouth.

Life history: Most are burrowers that feed on ants and termites, emerging after heavy rains. Most are chorus breeders. Amplexus in Gastrophryne olivacea is termed “glued” because the males possess specialized secretory cells on their ventral side that produce an adhesive substance for attachment to the back of the female.

Miscellaneous facts: This family contains more genera than any other Anuran family.

Species in lab: Gastrophryne sp. – Unidentified Narrow-mouthed Toad

What is one autapomorphy that you can use to identify this clade?

Pelobatidae – Spade Foot Toads Content and distribution: 3 genera, 11 species. North America, Europe, western and SE Asia. Only one species ( Scaphiopus holbrookii - not available in lab) is native to Massachusetts.

11 Biol 119 – Lab 4: Phylogeny & Anura Morphology: Pelobatids have a well-developed keratinous metatarsal tubercle, known as a spade , on each hind foot. Some have glandular skin, including parotoid glands. They are fairly squat animals, with short limbs, large eyes, and vertical pupils.

Examine some pelobatid specimens and identify the spade.

Life history: Many North American pelobatids are fossorial, emerging during the summer rains en masse to quickly eat and reproduce. They are explosive breeders, responding quickly to heavy rainfall and thunder. Tadpoles metamorphose very quickly (in just over a week in Scaphiopus couchii ). These frogs use their spades to dig burrows in which they aestivate most of the year. Although spadefoots are called “toads” because of their appearance, they are really fossorial frogs.

Species in lab: Scaphiopus couchii – Couch’s Spadefoot Scaphiopus sp. – Unidentified Spadefoot

What does “aestivate” mean? What sort of habitat would this be useful in?

Notice that some of the toads (Bufonidae) in lab have a keratinized structure on the hind foot, very similar to the spade of a spade foot toad. How do these structures differ between these two clades? (Hint: Check your field guide - it is a great source of information!)

Pipidae: Tongueless Frogs Content and distribution: 5 genera, 30 species. South America, sub-Saharan Africa.

Morphology: These frogs have no tongues. Their bodies are typically compressed dorsoventrally. Most have keratinous, clawlike fingertips and lateral line organs.

Life history: These frogs are fully aquatic. Some even call underwater. In some species, such as Pipa , there are elaborate courtship rituals which result in eggs sticking to the female’s back and ultimately they get enveloped by swelling of the skin. The eggs hatch in the female’s back either into tadpoles, or directly into adult form.

Miscellaneous facts: This family is not native to North America. However, Xenopus laevis is widely used in lab experiments as a model organism, and sometimes sold as a pet.

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Species in lab: Xenopus laevis – African Clawed Frog

“Ranidae”: True Frogs Content and distribution: 38 genera, 600+ species. Everywhere except southern South America, the Sahara Desert, and most of Australia. Five species are native to Massachusetts.

Morphology: Ranids typically have long legs, smooth skin, and pronounced webbing between the toes, and prominent tympana. Many also have dorsolateral folds. However, there are no unequivocal synapomorphies because they are likely paraphyletic.

Life history: These frogs typically live in and near lakes and ponds. Most are chorus breeders and lay eggs in water.

Miscellaneous facts: Many populations of ranids have been mysteriously declining in recent years. Some species are particularly susceptible to a chytrid fungus that keratinizes the skin, inhibiting cutaneous respiration. Some species have also been noted to be susceptible to developmental deformities, most likely associated with anthropogenic pollution. Although Lithobates catesbeianus is native to Massachusetts, it has been introduced in some states (for example, Arizona), where it out-competes native species.

Species in lab: Lithobates catesbeianus – American Bullfrog Lithobates clamitans - Green Frog Lithobates palustris - Pickerel Frog Lithobates pipiens – Northern Leopard Frog Lithobates sylvaticus – Wood Frog

For each species of Lithobates listed above in bold font, list one autapomorphy.

Do all of the species on display have dorsolateral folds? Do they vary in extent?

Why is "Ranidae" in quotation marks?

13 Biol 119 – Lab 4: Phylogeny & Anura REFERENCES Behler, J.L. and F.W. King. 1979. National Audubon Society field guide to North American reptiles and amphibians. Alfred A. Knopf, New York. Cannatella, D., L. Ford, and L. Bockstanz. 2000. Salientia. http://www.zo.utexas.edu/research/salientia/salientia.html. Conant, R. and J.T. Collins. 1998. A field guide to reptiles and amphibians - eastern and central North America. 3rd ed. Houghton Mifflin Co., Boston. Collins, J.T. 1997. Standard common and current scientific names for North American amphibians and reptiles. 4th ed. Society for the Study of Amphibians and Reptiles, Lawrence, Kansas. Crother, B.I. 2000. Scientific and standard English names of amphibians and reptiles of North America North of Mexico, with comments regarding confidence in our understanding. Society for the Study of Amphibians and Reptiles, Lawrence, Kansas. Degenhardt, W.G., C.W. Painter, and A.H. Price. 1996. Amphibians and reptiles of New Mexico. University of New Mexico Press, Albuquerque, New Mexico. Duellman, W.E. and L. Trueb. 1986. Biology of amphibians. McGraw-Hill, Inc., New York. McCord. R. 1998. Herpetology class notes. Pough, F.H., R.M. Andrews, J.E. Cadle, M.L. Crump, A.H. Savitzky, and K.D. Wells. 1998. Herpetology. Prentice-Hall, Inc., Upper Saddle River, New Jersey. Pough, F.H., R.M. Andrews, J.E. Cadle, M.L. Crump, A.H. Savitzky, and K.D. Wells. 2004. Herpetology. 3 rd ed. Prentice-Hall, Inc., Upper Saddle River, New Jersey. Stebbins, R.C. 2003. A field guide to western reptiles and amphibians. 3rd ed. Houghton Mifflin Co., Boston. Stebbins, R.C. and N.W. Cohen. 1995. A natural history of amphibians. Princeton University Press, Princeton, New Jersery. Zug, G.R., L.J. Vitt, and J.P. Caldwell. 2001. Herpetology: An introductory biology of amphibians and reptiles. 2 nd ed. Academic Press, San Diego, California.

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