1. Mariel Campbell Museum of Southwestern Biology, UNM Fall 2018
Polyphyly and Monophyly in the Family Linstowiidae: Cestode (Tapeworm) Parasites of Reptiles, Birds, Monotremes, Marsupials and Placentals Mammals
Mariel Campbell
Museum of Southwestern Biology, UNM
Fall 2018

2. A long time ago, as an undergrad here at UNM, I started a project to identify the parasites of mammals collected during a survey of Bolivia, I choose marsupials because they had an interesting biogeographic history. It turned out that they also had interesting parasites with an interesting biogeographic history. In particular, I focused on tapeworms in the genus Linstowia. At the time I originally did this research in the 1980s and early 1990s, I was limited to morphological analysis, so for this project my goal was to see if I could take another look at the systematics of this genus and its relatives by applying some of the techniques we are learning about in this course and hopefully, add in some genetic data.

3. The genus Linstowia belongs to a group of tapeworms found in reptiles, monotremes (echidna), marsupials, placental mammals, and birds. I am calling the group the family Linstowiidae, but I‘ll discuss in a bit whether that is really the case. While two of these genera (Oochoristica in reptiles and Mathevotaenia in mammals) are cosmopolitan, the majority of linstowiid taxa have a limited geographic and host range that includes many basal reptilian and mammalian lineages, such as African varanid lizards, South American amphisbaenids, the Australian echidna, and SE Asian tree shrews The pattern of association of many of these cestodes and their hosts suggests an ancient Pangean, Laurasian, or Gondwanian origin and distribution.

4. The genus Linstowia as presently described occurs in marsupials in South America and in marsupials and monotremes in Australia, Tasmania, and New Guinea. The disjunct distribution of this genus and the restriction to marsupial and monotreme hosts was cited by German systematics in the early 1900s (Zschokke, (1904, 1905) as evidence of common ancestry between Australian and New World marsupials, long before any understanding of continental drift.
Linstowia spp.

5. Did they take their tapeworms with them?
Marsupials evolved in Asia and North America, radiated in South America, and crossed Antarctica, to Australia million years ago (Nilsson et al 2010).
Did they take their tapeworms with them?
Current understanding based on morphological and genetic data is that marsupials evolved early in the diversification of mammals in the Northern Hemisphere, and then spread to South America and then to Australia via Antactica when the continents were still connected million years ago. All current marsupial taxa have been shown through retroposon data to be descended from South American ancestors (Nilsson et al 2010). Did they take their tapeworms with them?

6. Question 1: Does the Genus Linstowia represent a monophyletic group?
Hypothesis A: The genus Linstowia is monophyletic, with South American and Australian clades forming sister taxa. South American and Australian Linstowia species derive from a shared ancestor prior to the separation of the continents.
Hypothesis B:
The genus Linstowia is polyphyletic or paraphyletic. South American and Australian species of Linstowia are more closely related to other linstowiid taxa than to each other. Similarities between South American and Australian species of Linstowia are due to parallel evolution and/or independent acquisition from reptile ancestors.
Question: Does the Genus Linstowia represent a monophyletic group?
Hypothesis A: The genus Linstowia is monophyletic, with South American and Australian clades forming sister taxa.
South American and Australian Linstowia species derive from a shared ancestor prior to the separation of the continents.
Hypothesis B:
The genus Linstowia is polyphyletic or paraphyletic. South American and Australian species of Linstowia are more closely related to other linstowiid taxa than to each other. Similarities between South American and Australian species of Linstowia are due to parallel evolution and/or independent acquisition from reptile ancestors.

7. Morphological character matrix of 69 characters (28 multistate, 40 binary) for 26 Linstowiid taxa
For this analysis I was able to recover the morphological data set I used for my master’s thesis. This is a character matrix of 69 morphological characters for 26 Linstowid taxa. Dataset includes all members of the genus Linstowia plus additional sympatric representatives of Linstowid taxa from marsupials, placental mammals, and reptiles from both continents. There are 28 multistate and 40 binary characters; all characters were rescaled to give binary and multistate characters equal weight.
Taxa with more than one character state were interpreted as polymorphic. All multistate characters were treated as unordered using Fitch parsimony (Fitch, 1971). A maximum parsimony analysis was conducted using an heuristic search with TBR search algorithm. Trees were initially rooted with midpoint rooting to verify that ingroup taxa were monophyletic, subsequently, outgroup rooting was used with two species of Oochoristica from reptiles.
All multistate characters treated as unordered using Fitch parsimony. Taxa with more than one character state were interpreted as polymorphic. All characters were rescaled to give binary and multistate characters equal weight. Trees were initially rooted with midpoint rooting to verify that ingroup taxa were monophyletic, subsequently, outgroup rooting was used with two species of Oochoristica from reptiles.

8. Maximum Parsimony Analysis Results PAUP 4.0 26 taxa
Linstowia is not monophyletic based on this analysis of morphological characters.
Maximum Parsimony Analysis Results
PAUP 4.0
26 taxa
68 morphological characters
scaled for equal weights, base weight 1000
Heuristic Search, TBR, with 1000 Bootstrap replicates :
1 Tree
Length:
CI: 0.36
RI: 0.58
RC: 0.21
HI: .64
“Linstowia”=
Neotropical Marsupials
Oochoristica eremophila Australia
Atriotaenia New World skunks, raccoons
Paratriotaenia New World primates
Oochoristica antechini Australia
Mathevotaenia spp.
New world marsupials and edentates
Maximum Parsimony Analysis in Paup using heuristic search settings with TBR and 1000 bootstrap replicates yielded this consensus tree with a poor consistency index of 0.36.
Within each continent, the “Linstowia” spp appear to be monophyletic. It also appears that the species M. bivittata belongs in the clade with the South American Linstowia, an arrangement that had been proposed by the Russian taxonomist Spasskii in It appears from this analysis that these clades should be considered distinct genera.
The Australian taxa appear more basal, closer to taxa in reptiles in the New World and in Australia than they are to other members of the genus Linstowia;
The South American Linstowia are closer to parasites in the Australian genus Oochoristica from marsupials and to other South American linstowids in other genera than they are to members of the genus Linstowia in Australia.A major limitation, in retrospect, was not including representatives of all Linstowid genera in the analysis.
Other genera are also in question: Oochoristica and Mathevotaenia also polyphyletic; lumping of many lineages that don’t share a most recent common ancestor. These two genera are distinguished largely by whether the host is a mammal or a reptile, along with a few morphological characters.
In summary, the genus Linstowia is not a monophyletic group, the genus is polyphyletic. Based on this analysis we may not be looking a an ancient split between two closely related lineages., but independently acquired lineages more closely related to taxa in reptiles or other mammals.
“Linstowia” =Australian
Marsupials and Monotremes
Oochoristica : New World and Australian reptiles

9. Using the parasite phylogeny to map the hosts onto the continents, it would appear that this phylogeny supports an early divergence between SA and Australian taxa followed by a recolonization of South America with Australian-like forms. It also predicts that there are at least two major clades of Linstowids in marsupials and other mammals in both Australia and South America. But we are missing too many taxa to really understand relationships in this group, and this hypothesis should be retested using molecular genetics.

10. The next step was to find sequences
The next step was to find sequences. Unfortunately, the original Bolivian material I worked with, while deposited in a museum, had been mostly preserved in formalin. A GenBank search for Linstowiid genera yielded exactly one sequence, a partial 28S ribosomal gene from the species Mathevotaenia symmetrica from an African rodent. Since, I don’t have enough genetic material to use molecular methods to answer my first question about relationships of species in the genus Linstowia, I decided to see if I could use this sequence to answer questions at the family level.

11. What is the status of the Family Linstowiidae?
Question 2:
What is the status of the Family Linstowiidae?
Despite elevation to family by Spasskii 1951, the “Family” Linstowiidae has not been recognized in the literature.
Placed historically in the Family Anoplocephalidae.
Are Linstowiid cestodes a subfamily of Anoplocephalidae or a separate family?
E.g. Do they share a most recent common ancestor with anoplocephalids or with other taxa?
With this one sequence I can address Question 2:
What is the status of the Family Linstowiidae?
Placed historically in the Family Anoplocephalidae.
Despite elevation to family by Spasskii 1951, the “Family” Linstowiidae has not been recognized in the literature.
Are Linstowiid cestodes part of the family Anoplocephalidae or a separate family?
E.g. Do they share a most recent common ancestor with anoplocephalids or with other taxa?

12. Relationship of Linstowid cestodes to Family Anoplocephalidae and other Families in Order Cyclophyllidea:
Methods:
1) Search GenBank: single linstowid sequence (Mathevotaenia symmetrica, 28S)
Blasted sequence against other Cestoda
Selected representatives of other families in Order Cyclophyllidea with 28S sequence data;
Choose representatives with lowest E-scores and greatest sequence overlap
Selected outgroup taxa in Order Diphyllobothridea
Downloaded and aligned sequences of 46 cestode taxa
Compared alignments by Muscle and Clustal Omega
Trimmed and untrimmed sequences
5) Maximum Parsimony analysis in Paup 4.0
RaxML analysis
Bootstrapped 1000 replicates
Analyzed resulting trees
Methods for answering this question:
Searched GenBank: for linstowid sequences: found (Mathevotaenia symmetrica, 28S), the only sequence for this group
Blasted sequence against other Cestoda
Selected representatives of other families in Order Cyclophyllidea with 28S sequence data;
Choose representatives with lowest E-scores and greatest sequence overlap
Selected outgroup taxa in Order Diphyllobothridea
Downloaded and aligned sequences of 46 cestode taxa
Compared alignments by Muscle and Clustal Omega
Trimmed and untrimmed sequences
5) Maximum Parsimony analysis in Paup 4.0
RaxML analysis, bootstrap replicatesared resulting trees

13. Dataset: 46 taxa; sequence alignment 5300bp trimmed to 2375
This is just a glimpse of the kind of alignment I got at the family level with these very different sequences. My initial alignment of 46 taxa covered 5800 bp but had to be trimmed. I initially used Mega and aligned with Muscle, and got poor alignments because of some divergent taxa and only partial sequence alignment for all of the taxa. I trimmed to 2375 bp and removed the divergent, poorly aligned taxa and realigned using Clustal omega and that seemed at least visually to give a much better alignment Muscle didn’t work as well; not as good with many loose ends (N/C terminal extensions) as Clustal in this case.
Dataset: 46 taxa; sequence alignment 5300bp trimmed to 2375

14. PAUP Maximum Parsimony Analysis of 46 Cestode Taxa Using 28S nuclear rRNA
Outgroup rooting
Trimmed alignment
1000 BS replicates
Heuristic Search
Default settings
Tree Length 6008
CI RI RC HI
Anoplocephalidae
Parsimony Analysis of 46 Cestode Taxa using 28S ribosomal RNA using PAUP 1000 BS , Diphyllobothrium as outgroup; Family Classification is the first two capitalized letters. Red shows the family Anoplocephalidae as currently defined and green shows the branch containing the Linstowiid query species Mathevotaenia.
1) Supports monophyly of most major families of Cyclophyllideans, with high BS support (100) for most families; but basal branching is not resolved
Shows a clustering of the families Anoplocephalidae and Hymenolepididae, with one basal Anoplocephalid outside the clade
The Linstowid species Mathevotaenia symmetrica shows low bootstrap resolution but does not group with the Anoplocephalids; however it does group with environmental sample from carrion beetles that showed up during the Blast search. This is very interesting as the life cycle of this group is unknown, and these results suggest for the first time the possibility of carrion beetles as an intermediate host of the larval cestodes in this group
Note position of Linstowiids clustering with other unresolved taxa in this group – PA, DA, TA, CA families – rather than within the Anoplocephalidae
Linstowiidae

15.
RaxML Analysis of 46 Cyclophyllidean Cestode Genera using nuclear ribosomal 28S
100 Bootstrap replicates
GTR Gamma+I
TBR Heuristic
1 Tree
-ln L = 29071
AIC = 58329
The RaxML analysis had better resolution, with strong bootstrap support for many of the major families and moderate support for basal branches. This analysis also showed a similar pattern to the parsimony analysis, with Anoplocephalidae and Hymenolepididae grouping together as sister taxa, and the Linstowid taxon Mathevotaenia on a more basal branch showing strong identity to the carrion beetle environmental sample.
There is moderate support for the positioning of Mathevotaenia just above the basal families in grey and decidedly outside of Anoplocephalidae.

16. The only previous molecular phylogenetic work I was able to find that included Linstowid taxa was a 2017 publication: Tapeworms from the Vertebrate Bowels of the Earth. As part of a global survey of tapeworm taxa over 9 years, the authors recovered a single Linstowid cestode in the genus Oochoristica from a colubrid snake in Iran.
Bayesian analysis of nuclear 28S, 18S, mitochondrial 16S and COI genes placed Linstowiidae as a more basal group outside of the Anoplocephalidae/Hymenolepididae clade, clustering with Davaineidae, Taeniidae, Paruterinidae, Meta (Dilepididae), Catenotaeniidae.
These results are similar to the results I obtained from the Mathevotaenia and environmental 28S sequences samples from GenBank analyzed by RaxML.
The conclusions from both of these studies are that the cestodes in this group should be a separate family from the Anoplocephalidae. This corresponds to a recommendation by Spasski 1951, a Russian morphological taxonomist whose work was largely ignored in the West.

17. Summary
Morphological analysis suggests that the genus Linstowia does not represent a monophyletic group sharing a common ancestor dating from prior to the separation of the continents.
Based on this analysis, the genus represents separate South American and Australian lineages that should be split into separate genera. These lineages may have been acquired separately from parasites of reptiles.
Based on molecular analysis of two sequences, Linstowiid cestodes are evolutionarily distinct and should be a separate family distinct from cestodes in the Family Anoplocephalidae.
This study provides evidence that carrion beetles may be a potential intermediate host for the Family Linstowiidae.
The group is very poorly known with many interesting, ancient, and potentially informative lineages that can be examined to study host and parasite evolutionary and phylogeographic patterns. But we need molecular samples.
Summary
Morphological analysis suggests that the genus Linstowia does not represent a monophyletic group sharing a common ancestor dating from prior to the separation of the continents., but instead represents separate South American and Australian lineages that should be split into separate genera. If this is the case, these lineages may have been acquired separately from parasites of reptiles, and similarity in morphology could be due to parallel evolution. But we need molecular data.
Based on molecular analysis of only two sequences, Linstowiid cestodes appear to be evolutionarily distinct and should be a separate family distinct from cestodes in the Family Anoplocephalidae.
The life cycle of this group of tapeworms is unknown. However, based on the GenBank environmental sample that clustered with the Mathevotaenia sequence, there is now evidence that carrion beetles (Coleoptera: Silphidae)may be a potential intermediate host.he group includes ma
However, in order to understand anything about the interesting taxa in this family we are going to need a lot more genetic samples.
A number of these taxa can be found in New Mexican reptiles and mammals, so please let me know if you are collecting any of these taxa as I would be very interested in recovering their parasites.

18. References
Beveridge, I. "The genus Linstowia Zschokke, 1899 (Cestoda: Anoplocephalidae) in Australian mammals with the description of a new species, L. macrouri." Systematic Parasitology 5.4 (1983):
Caira, Janine N., and Kirsten Jensen. Planetary biodiversity inventory (2008–2017): Tapeworms from vertebrate bowels of the earth. Natural History Museum, University of Kansas, 2017.
Janicki, C, . VON Studien an Siugetiercestoden. Zeitschrift fir Wissenschaftliche Zoologie 81:
Kaltenpoth, Martin, and Sandra Steiger. "Unearthing carrion beetles' microbiome: characterization of bacterial and fungal hindgut communities across the S ilphidae." Molecular Ecology 23.6 (2014):
Nilsson MA, Churakov G, Sommer M, Tran NV, Zemann A, et al. (2010) Tracking Marsupial Evolution Using Archaic Genomic Retroposon Insertions. PLOS Biology 8(7): e  
Pais, F. S., Ruy, P. C., Oliveira, G., & Coimbra, R. S. (2014). Assessing the efficiency of multiple sequence alignment programs. Algorithms for molecular biology : AMB, 9(1), 4. doi: /
Schiewe, J. (28 July 2010). "Australia's marsupials originated in what is now South America, study says". Los Angeles Times. Archived from the original on 1 August Retrieved 1 August 2010.
Schmidt, G, . D Handbook of tapeworm identification. CRC Press, Boca Raton, Florida6
Spasskiĭ, Alekseĭ Andreevich. Anoplocephalate tapeworms of domestic and wild animals. Vol. 1. Academy of Sciences of the USSR, 1951 ie Jerusalem, Israel Program for Scientific Translations, 1951.
Wiens, John J. "Character analysis in morphological phylogenetics: problems and solutions." Systematic Biology50.5 (2001):
Zschokke, F., Neue Studien an Cestoden aplacentaler Saugethiere. Zeitschrift fur Wissenschaftliche Zoologie 65:
Zschokke, F. 1904a. Die Darmcestoden der amerikanischen Beuteltiere. Centralblatt fur Bakteriologie, Parasitenkunde und Infektionskrankheiten, 1st Abt. 36:
Zschokke, F. 1904b. 2. Die Cestoden der siidamerikanischen Beuteltiere. Zoologischer Anzeiger 27:

19. Acknowledgments:
Dr. Scott L. Gardner
Bolivia Field Crews