Phylogenetics of
Early-Diverging Cacti
by
Ruth Kirkpatrick, Abby Moore, Bianca Knoll, Vicente Garcia, Andy
Murdock, Michael Park, and Anna Larsen
Maihuenia
Maihuenioideae
Peresk
ia
Pereskioidea
e
TRADITIONAL
CACTACEAE
Opuntia
SUBFAMILIES
Opuntioideae
Cactoideae
Opunti
a
Echinocactu
s
Cereus
THINGS TO DO BEFORE LAB
1. Read “Introduction to Cladistic Analysis” (p. xxx) and the Introduction to the Cladistics of EarlyDiverging Cactus Lineages (p. xxx) before coming to lab. Your GSI will assume that you know
how systematists use cladograms to test phylogenetic hypotheses.
2. Make sure that you’re familiar with the terms and concepts listed below. Consult your lecture
notes, textbook, or this lab manual about any items that are new or unclear to you before coming
to lab.
adaptive radiation
analogous traits
cladistics
cladogram
convergent evolution
derived
homologous
homoplasy
ingroup
lineage
monophyletic
outgroup
paraphyletic
phylogenetics
phylogeny
polyphyletic
relictual
systematics
taxon (plural taxa)
vestigial
Phylogenetics of Early-Diverging Cacti
A teaching module and laboratory exercise for CIPRES
INTRODUCTION
The Cactus Family (Cactaceae) includes approximately 1500-1800 species distributed
throughout the New World, with centers of diversity in the warm and arid regions of North and South
America. Cacti are especially abundant in the southwestern United States and Mexico, eastern Brazil,
and the slopes of the Andes. Humans have used a number of species in various ways for about 12,000
years.
Cacti are well known for their remarkable morphological and physiological adaptations to
drought, and represent one of the world’s most spectacular desert radiations. Many members of the
cactus family lack photosynthetic leaves and instead photosynthesize with their green and succulent (i.e.
water-storing) stems. In contrast, families of plants adapted to moister environments photosynthesize
with their more “typical” leaves and lose a great deal of water through their stomata via transpiration.
In addition to succulent stems, cacti also have a water-conserving type of photosynthesis called CAM
metabolism that involves taking carbon dioxide in through their stomata at night when it is cool and
transpiration is low, and using light energy during the day to drive the sugar building phase of
photosynthesis. Thus, most cacti are adapted to living in dry environments in at least three ways: (1) by
photosynthesizing with their stems; (2) by storing water, when it is available, in deeper tissues of their
stems; and (3) by using a water-conserving form of photosynthesis. There are also many other features
of cacti that are thought to be adaptive to xeric environments.
Traditionally, the great majority of cacti were divided into two major groups, the Opuntioideae
(“Prickly Pear” and “Cholla” cacti) and Cactoideae (“Columnar” and “Barrel” cacti) subfamilies. Most
members of these two groups are considered “typical” cacti and are recognized by their stem
succulence, vestigial or ephemeral leaves (or complete absence of green leaves), well-developed
photosynthetic stems with CAM metabolism, specialized short stems called areoles (see below), and
residence in hot and dry environments. The Opuntioideae is characterized by the presence of glochids
(barbed hairs) in the areoles, while the Cactoideae (i.e., Columnar and Barrel cacti) is distinct in its
complete lack of leaves.
Two small genera, Maihuenia and Pereskia, make up the remaining two groups of cacti (the
subfamilies Maihueniodeae and Pereskioideae, respectively). These two genera have been grouped
together based on the shared absence of many of the “typical” cactus characters listed above. Both
Maihuenia and Pereskia produce leaves that perform the greater part of photosynthesis for these plants.
Maihuenia consists of two species of highly specialized cushion plants that live in cold, dry regions of
Patagonia and south-central Chile. These cushion plants grow in very dense stands of short stems with
leaves crowded at the tips. Pereskia species are widely distributed in the Caribbean and Central and
South America in a range of warm and seasonally dry forest habitats. Pereskia species are often
interpreted as “relictual cacti,” and are used as a general model (the “Pereskia Model”) for the
“ancestral” condition from which the highly specialized morphology and physiology of typical cacti
arose. Pereskia live in relatively mesic environments and have broad and flat leaves; areoles that
produce spines and photosynthetic leaves; and non-photosynthetic, non-succulent stems.
Although these four lineages of cacti have distinct characteristics they also share characters that
unite them as members of the Cactaceae. For example, all members of the cactus family are
characterized by the presence of spines (highly modified leaves and stems) on their stems. Therefore,
PLEASE BE CAUTIOUS OF SPINES WHEN HANDLING THE CACTI!
Another characteristic of all the members of the Cactaceae is the production of two distinct types
of stems called “long shoots” and “short shoots”. A long shoot is simply a stem with long internodes.
Short shoots are stems with much shorter internodes. These terms are applied only in plants in which
internode length is clearly bimodal and both long and short shoots are present. The short shoots of cacti
have especially short internodes and are called areoles. Areoles produce: spines, new long shoots
(a.k.a.: branches), glochids, and flowers. See the labeled plant body and cactus body diagrams below
(Figures 1and 2).
The combination of floral characters of the Cactaceae flower is also distinctive. Their flowers
are bisexual (i.e. they have both male stamens and female pistil (several fused carpels), have radial
symmetry, and possess many showy and spirally arranged tepals (sepals and petals that look alike).
Their flowers also have inferior ovaries (see ovary positions below and in glossary) that are deeply
recessed into the apex of modified stems, so the outer portion of a cactus ovary is covered with spinebearing areoles. See the labeled diagram of a typical cactus flower below.
Because of these shared characters, all cacti have historically been considered
closely related and the Cactaceae has been formally recognized as a family since the
1780s. There have been disagreements about the closest relatives of the Cactaceae.
Recent studies based on DNA sequence data suggest that the Portulacaceae (The Purslane
Family) is the sister group of the Cactaceae. The presence of succulent tissues and
CAM metabolism are two characteristics found in members of both the Cactaceae and
Portulacaceae and are thought to have been inherited from their common ancestor.
Portulacaceae lacks many of the other “typical” cactus characteristics. On the basis of
this hypothetical sister group relationship (i.e., Cactaceae and Portulacaceae s.l. are both
monophyletic and each is the other’s nearest relative), several species of the
Portulacaceae will be used as the outgroup taxa in your phylogenetic analysis of the
Cactaceae. Outgroup selection is a challenging aspect of cladistic analysis and is
simplified for you here. Outgroup character states are used to determine the direction of
evolutionary change in cladistic analysis. The particular character state in an outgroup
taxon is chosen as the ancestral condition and differences in homologous character states
in the ingroup are considered to be the derived condition(s).
While the “Pereskia Model” (see above and glossary) has been useful for
thinking about the evolution of more derived cacti, it also downplays some potentially
important ways in which Pereskia species differ from one another. An alternative
hypothesis for Pereskia is supported by studies that have emphasized the substantial
ecological, morphological, and anatomical diversity found within Pereskia and between
Pereskia and Maihueniodeae. The cladograms below depict several possible hypotheses
of relationships among the members of the Cactaceae. There are many more
possibilities.
Cladogram A: Traditional view of all subfamilies monophyletic with Pereskia the
earliest diverging lineage and Cactoideae and Opuntioideae most derived.
Cladogram B: Pereskia paraphyletic and sister to monophyletic Maihueniodeae,
Cactoideae, and Opuntioideae.
Cladogram C: Monophyletic Opuntioideae, Maihueniodeae, and Cactoideae all nested
within a paraphyletic Pereskia.
Cladogram D: Monophyletic Opuntioideae, Maihueniodeae, and Cactoideae all nested
within a polyphyletic Pereskia.
Each of the above cladograms depicts different relationships among the living
members of the Cactaceae. Each tells a different story about the evolution of
morphology and physiology as well as the biogeographic history and ecological
adaptations among members of the family. By obtaining the “true” phylogeny, much
insight can be gained and further investigations can be made into the evolutionary history
of this diverse and interesting plant family.
The ability to infer early events in the evolutionary history of the Cactaceae
depends on resolving two outstanding problems in cactus phylogeny:
(1) Whether Pereskia is monophyletic, and
(2) How Pereskia species are related to the rest of the cacti.
Recall from the “Introduction to Cladistics” section in your lab manual that a
cladogram is a branching diagram, where the taxa under study are represented at the tips
of the branches and the hypothetical ancestors at the nodes. A cladogram is thus a
hypothesis of the relationships among the group of taxa under study. A monophyletic
group (or clade): (1) consists of an ancestral species and all of its descendant species,
and (2) is based on the common possession of shared-derived characters
(synapomorphies). Ideally, all named groups of organisms are monophyletic.
A paraphyletic group consists of an ancestral species and some, but not all, of its
descendants. Members of paraphyletic groups are often recognized by their shared
possession of many primitive characters (symplesiomorphies). Thus, more derived taxa
that lack many ancestral characters, even though they are descended from the common
ancestor, are not grouped with their more primitive looking relatives and their absence
results in a paraphyletic group. For example, someone could redefine plants to include
only things with large, flat, and green leaves. This would be a paraphyletic taxon
because most cacti would be excluded even though they too are descended from the
common ancestor of leafy green plants.
Polyphyletic refers to a grouping of species derived from two or more different
ancestral forms. Polyphyletic groups thus include two or more distantly related taxa, but
exclude their common ancestor and most of the descendants of that common ancestor. In
other words, polyphyletic groups are constructed by taking branches from distantly
related parts of the tree of life. They are often characterized by the possession of at least
one “similar” character that evolved independently (e.g., by convergent evolution) in
each member of the group. These convergent characters often perform similar functions
and cope with similar challenges in the environments in which these organisms live.
These convergent characters are analogous (as compared to homologous characters that
are similar because they are inherited from a common ancestor). Analogous characters
can be identified on a cladogram and in cladistic terms are called homoplastic characters
or homoplasies. Homoplastic characters can make cladogram construction more difficult,
but their discovery during cladistic analyses may suggest interesting adaptive scenarios.
For example, a polyphyletic grouping of “fast swimmers” with streamlined
hydrodynamic bodies may include: ichthyosaurs (reptiles), sharks (cartilaginous fish),
tuna (bony fish), dolphins (marine mammals), and penguins (birds) (see also Figure 40.2
in Campbell, Biology 7th ed.).
Systematics is the classification of living organisms into hierarchical series of
groups emphasizing their evolutionary relationships (a.k.a. phylogeny). Ideally, the
classification and names of organisms reflect their evolutionary relationships. Many
systematists make use of cladistic methods in their attempts to generate classifications
that reflect “true” evolutionary relationships. Cladistic methods use recency of common
ancestry as the criterion for grouping taxa and generating phylogenetic hypotheses
inferred from the resulting cladograms. These hypotheses of relationship are then used as
a basis for classification.
YOUR GOAL
In this lab, you and your teammates will conduct a cladistic analysis of cactus
species that represent a cross-section of the vegetative morphological diversity found in
the four traditional subfamilies of Cactaceae: Pereskioideae, Maihueniodeae, Cactoideae,
and Opuntioideae. Your team will prepare a laboratory report that describes and explains
this lab’s questions, research methods, and results.
The traditional classification of the Cactus Family (Cactaceae) is based on its
distinctive morphologies, some of which were described above. The species available for
you to examine in lab today are listed below in their traditional subfamily groupings.
Your job will be to determine if the traditional classification reflects the “true” phylogeny
of the Cactaceae. You and your lab group will use the results of your cladistic analysis of
morphological and molecular sequence data to explain if, how, and why the traditional
sub-family groupings of the Cactaceae reflect the “true” phylogeny or not. Your team
will also interpret and discuss the implications of the phylogeny that you generate and the
evolutionary history of characters, habitat, and distribution of members in the Cactus
Family.
OUTGROUP = close relatives of Cactaceae
Portulacaceae (Purslane Family)
Anacampseros telephiastrum (“Pan American Love Plant”)
Portulaca pilosa (“Pink Purslane”)
INGROUP = Cactaceae (Cactus Family)
TRADITIONAL SUBFAMILIES
Cactoideae
Opuntioideae
Cereus sp. (“Cereus”)
Austrocylindropuntia subulata (“Cane Cholla”)
Echinocactus sp. (“Hedgehog Cactus”)
Opuntia longispina (“Prickly Pear”)
Pereskiopsis porteri (“Alcajer”)
Quiabentia verticillata
Tephrocactus glomeratus (“Paper Spine Cactus”)
Maihueniodeae
Maihuenia poeppigii (“Chupa Sangre”)
Pereskioideae
Pereskia aculeata (“Barbados Gooseberry”)
Pereskia bleo (“Wax Rose”)
Pereskia grandifolia (“Rose Cactus”)
HOW TO BUILD A CHARACTER DATA MATRIX AND THEN COVERT THIS
INFORMATION INTO A TREE.
The process of phylogenetic analysis consists of first assembling a data matrix
and then inferring a phylogenetic tree from that matrix. The tree is basically just a rerepresentation of the data matrix with no value added. The elements of the data matrix
are (1) the rows (Who are the taxa?), (2) the columns (What are the characters?), and (3)
the individual entries (What are the character states?). Ideally, characters are
homologous features that vary independently among the taxa. Character states are
discrete conditions of a character and potential phylogenetic markers.
Remember that analogous characters (homoplasies) resulting from convergent
evolution and other processes that distort the phylogenetic signal can make tree building
challenging. The only weapon against this distortion is the use of many independent
sources of information (characters) that are, as best as can be determined, not affected by
the same biasing processes. The trees you build from the character matrix data may not
be completely resolved (contain only two branches at every node) and supported by
shared derived characters. Unsupported branches may need to be collapsed with several
branches arising from a single node (an unresolved node or polytomy).
I. Venn Diagram Method of converting a Data Matrix into a Cladogram,
“WITH FIGURES”:
First, What is a Venn diagram? Venn diagrams are graphical representations of sets and
subsets of data that are made by encircling data points that have shared characters in
nested boxes or circles that then depict relations within the data set. Venn diagrams are
used here to group clades and subclades to show relationships. Branches are then drawn
below the Venn diagram nested boxes to reflect these nested relationships in the form of
a cladogram.
II. Cladistic Analysis using Venn Diagram Method, shown here as a flow diagram
of four steps analyzing the 3 Domains of Life: Bacteria, Archaea, and Eukarya.
III. HOW TO:
Venn-Diagram Method of converting a Data Matrix into a Cladogram, “IN
WORDS”:
1. Complete your character analysis by determining the character state of the taxa in the
matrix for each of the characters selected and listed in the matrix.
2. Determine if a particular character state is derived or primitive, based on comparisons
to the outgroup’s character state. (See “Outgroup Comparison” in the “Introduction to
Cladistics” section of your lab manual.) The outgroup’s character state is considered the
primitive condition.
3. In each column of your matrix, encircle only the groups of shared derived characters
(synapomorphies). This will help you to identify monophyletic groupings within your
matrix.
4. Identify taxa subsets (= subclades and clades) based on their shared derived characters
(synapomorphies).
5. Place subclades and their larger (sub-)clades into “nested” boxes (see Venn Diagram
above).
6. Draw cladogram branches below taxa boxes to reflect relationships depicted by these
nested boxes (e.g., clades and subclades).
7. Label cladogram tips with taxa names and branches with supporting shared derived
characters (synapomorphies).
PERFORMING A CLADISTIC ANALYSIS OF THE CACTUS FAMILY
Set-up
Obtain for your lab group one set of potted cactus (ingroup) and Portulacaceae
(outgroup) species and one copy of the supplemental photographic library of these
species.
Hypothesis formation
Based on the traditional classification of the Cactus Family listed above and described in
the introduction, draw a cladogram that best represents this “traditional” hypothesis of
relationship among these taxa. Develop clear and concise alternative hypotheses about
the evolutionary relationship among the four lineages of the Cactus Family. Make
predictions about the results you would expect under each hypothesis. You will include
these hypotheses, predictions, and results in your lab report.
I. Step-by-step cladistic analysis of the Cactus Family using morphological data:
Use the charts and boxes on the following pages to carry out a cladistic analysis of the
cactus species. Because the identification of informative characters in morphologically
complex structures is often very difficult and time consuming, characters have been
selected for you. Labeled diagrams, observations of living plants, and your GSI will help
you interpret the morphology and determine the character states for each cactus and
outgroup species.
Step 1: Group the pots of ingroup taxa together for ease of examination.
Step 2: Group the pots of outgroup members together.
Step 3: Examine the morphology and select character states of the ingroup taxa.
Step 4: Examine the morphology and determine the character states of the outgroup
taxa.
Step 5: Determine whether each character state is ancestral (a plesiomorphy) or
derived (an apomorphy) (“O” indicates a ancestral and “1” or “2” represents various
derived character states). These character states are determined by comparison with the
outgroup taxa.
Many characters, character states, and character assignments are provided for you
in the following morphological matrix. Work with your lab group members and fill in
the matrix by determining the character states of all taxa for the characters provided.
Step 6: Circle shared derived characters in each column to recognize and form clades.
Step 7: Convert data matrix into Venn Diagrams (nested boxes) of subclades and clades
based on shared derived characters.
Step 8: Draw and connect branches below the Venn diagram nested boxes and transform
taxa relationships into a branching diagram (cladogram) with each taxon at the tips.
Step 9: Redraw your cladogram and label each branch tip with ingroup and outgroup
names. Add characters by labeling branches that are supported by shared derived
characters (synapomorphies) and uniquely derived characters (autapomorphies).
II. Now perform a cladistic analysis of the already filled in DNA sequence data
matrix:
The entire length of the molecular sequence data generated from the original research of
Edwards et al. (2004) has been drastically “pruned” of species not used in this lab and of
many of the molecular sequence characters. The original data matrix, in its entirety, is
stored on the TreeBASE website (http://www.treebase.org/treebase/).
Here is the pruned DNA sequence matrix for you to analyze. It contains a very small
subset of only informative characters. This will make your cladistic analysis of molecular
sequence data easier.
Perform steps 6 – 9 with this molecular sequence data.
Venn diagrams & Tree Drawings:
III. Now perform a cladistic analysis on a combined morphological and molecular
data matrix.
Perform steps 6 – 9 with your combined morphological and molecular data matrices.
Questions to consider:
1. Are your three cladograms (1st based only on morphology, 2nd based only on
molecular data, and 3rd based on combined morphological and molecular data) the same
or different? What do the three cladograms tell you about the history of the Cactaceae?
2. Is Pereskia monophyletic, paraphyletic, or polyphyletic?
3. Can you identify any homoplastic characters and/or any paraphyletic or polyphyletic
groups?
4. Can you suggest and describe any adaptive hypotheses based on analogous
(homoplastic) characters and/or any polyphyletic taxa?
5. Why do you think the morphology of Maihuenia poeppigii is so very different from its
closest relatives? What might the retention of leaves suggest about possible functions
and/or adaptive scenarios?
YOUR ASSIGNMENT:
As a team, prepare a lab report that includes the following parts:
1. Title (few words to one sentence)
Identify the problem or question that you investigated in this lab.
2. Introduction (one page)
Describe your problem, explain its significance, list your alternative hypotheses
about the evolutionary relationship between the four lineages of Cactaceae, and describe
the results of a cladistic analysis that you would expect under each hypothesis.
3. Materials and Methods (one sentence to several paragraphs)
Just cite the relevant pages in this lab manual. However, if you used different
materials or deviated from the procedure given here, then include detailed descriptions of
these changes.
4. Results (one to two pages)
Simply insert the set of data matrices with circled clades, Venn diagram nested
boxes, and drawn and labeled cladograms into your report.
5. Discussion (one page)
Interpret your results and state your conclusions (indicate whether or nor your
results confirm your predictions, and whether they support or negate each of your
hypotheses). State the significance of your results. List any weaknesses with the method
that you followed, and tell how they may have affected your results.
Each team member will receive the same grade. Your GSI will announce the due-date
for this assignment.
Literature utilized in the development of this lab exercise:
Anderson, E. F. 2001. The Cactus Family. Portland, Oregon: Timber Press Inc.
Campbell, N. A., and J. B. Reece. 2005. Biology, 7th edition. San Francisco, California:
Pearson, Benjamin Cummings.
Edwards, E. J., R. Nyffeler, and M. J. Donoghue. 2005. Basal Cactus Phylogeny:
Implications of Pereskia (Cactaceae) paraphyly for the transition to the cactus life form.
American Journal of Botany 92(7): 1177-1188.
Esau, K. 1977. Anatomy of seed plants. 2nd ed. New York: John Wiley and Sons.
Foster, A. S. and E. M. Gifford, Jr. 1974. Comparative morphology of vascular plants.
2nd ed. San Francisco: W.H. Freeman and Company.
Green, Adam. Barrel cactus (drawing). Retrieved 11/22/05 from http://www.sketchup
models.com/Merchant2/merchant.mvc?Screen=PROD&Product_Code=0000246_agreen
&Category_Code=mpd&Product_Count=0.
Garchette, F. Maihuenia poeppigii (photo). Retrieved 11/25/05 from Au Cactus Francophone, http://www.cactuspro.com/photos.php?action=afficher&id=1337.
Hickey, M. and C. King. 2000. The Cambridge illustrated glossary of botanical terms.
Cambridge: Cambridge University Press.
Judd, W. S., C. S. Campbell, E. A. Kellogg, P. F. Stevens, and M. J. Donoghue. 2002.
Plant Systematics, A Phylogenetic Approach, 2nd edition. Sunderland, MA: Sinauer
Associates.
Mishler, B. D. 2004. The logic of the data matrix in phylogenetic analysis. Pp. 57-70, in
Parsimony, Phylogeny, and Genomics, ed. Victor A. Albert. Oxford: University Press.
Nyffeler, R. 2002. Phylogenetic relationships in the cactus family (Cactaceae) based on
evidence from trnK/matK and trnL-trnF sequences. American Journal of Botany 89(2):
312-326.
Zomlefer, W.B. 1994. Guide to flowering plant families. Chapel Hill: University of
North Carolina Press.
Contributors:
Ruth Kirkpatrick: project leader, lab exercise and glossary composition, final editor and
lab exercise design.
Abby Moore: data matrix composition, lab exercise and glossary editing and
composition.
Bianca Knoll: instructional minigrant composition, lab exercise and glossary editing and
composition, lab exercise and glossary illustrations.
Andy Murdock: acquisition of live plants and lab exercise editing.
Vicente Garcia: Cactus Biogeography supplemental exercise and Cactus Photo Library.
Michael Park: initial Venn diagram design and key.
Anna Larsen: instructional minigrant composition and lab exercise editing.
Dr. Dean Kelch: acquisition of live plants and culture consultant.
Drs. Glenys Thomson, Montgomery Slatkin, John Latto, and Brent Mishler: faculty
sponsors and project reviewers.