Whole-collection COI barcoding and species delimitation of Neopilionidae () reveal need for family-wide taxonomic revision Jonathan MacDougall1, Kate Sheridan2, Christina J. Painting3, Gregory I. Holwell3, Abel Pérez-González4, Ricardo Pinto-da-Rocha5, Gustavo Hormiga1, Gonzalo Giribet2 1 Department of Biology, The George Washington University, Washington, DC 20052, USA 2 Museum of Comparative Zoology, Harvard University, Cambridge, MA 01238, USA 3 Te Kura Mātauranga Koiora, School of Biological Sciences, Te Whare Wānanga o Tāmaki Makaurau, University of Auckland, New Zealand 4División de Aracnología, Museo Argentino de Ciencias Naturales “Bernardo Rivadavia” – CONICET, Av. Ángel Gallardo 470, C1405DJR Buenos Aires, 5Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, Caixa Postal 11461, 05422-970 São Paulo, SP, Introduction Introduction The harvestmen of the family Neopilionidae are distributed across temperate Gondwana, in Chile, Argentina, southern Brazil, South Africa, and New Zealand. In New Zealand, neoplionids are represented by 27 described species in seven genera1. We have assembled an extensive dataset of COI sequence data of neopilionids to assess species limits in the group. Several neopilionid species are highly polymorphic in morphological characteristics such as the size and shape of male chelicerae2, while others are identified from female specimens that lack many defining characteristics, creating difficulties in describing species and identifying specimens. In order to determine species boundaries in our dataset, we used the multi-rate Poisson Tree Process (mPTP), which allows for different species to have evolved at different rates and yields more accurate delimitations at a faster rate than previous programs3. Methods Methods • Sequenced COI fragment for 689 specimens

• 7 outgroup terminals: two Dyspnoi taxa (Trogulus and Ischyropsalis genera) and representatives from all five families

• Sequences aligned using MAFFT v.74

• Tree inferred using IQ-TREE5 on CIPRES Science Gateway6

• Species delimitation run with mPTP3 using 4 MCMC runs of 1,000,000 generations

P. cheliferoides, P. listeri, male male (Photo, G (Photo, G Giribet) Giribet)

P. listeri, female P. listeri, male (Photo, G (Photo, G Giribet) Giribet)

Discussion Discussion • Well supported species clades, poorly supported genera and backbone relationships • Pantopsalis and Forsteropsalis in need of revision • Polyphyletic genera • Fewer species in Pantopsalis, more species in Forsteropsalis than previously thought • Evidence for synonymizing P. listeri and P. cheliferoides • Evidence for synonymizing Thrasychiroides and Thrasychirus • Monospecific genera (Mangatangi, Templar) show need for new species descriptions

Acknowledgments Acknowledgments This work was supported by a US National Science Foundation grant to G. Hormiga, PI at GWU, G. Giribet, PI at Harvard University, S. Boyer PI at Macalester (“Collaborative Research: The Opiliones of New Zealand: Revisionary synthesis and application of species delimitation for testing biogeographic hypotheses)”(DEB 1754289, 1754278, and 1754262). We would like to thank members of the Hormiga Lab for providing support and advice. ML tree showing bootstrap support for 51 mPTP delimited neopilionid species. Colored circles indicate bootstrap support for phylogenetic inference; all nodes received ≥0.95 bootstrap support from mPTP as a speciation event. Color-coded References References according to putative identity: Thrasychirus, Thrasychiroides, New Caledonia 1. Taylor CK. (2013) Zookeys 263: 59–73. 2. Fernández R, Vélez S, Giribet G. (2014) Invertebrate Systematics 28, 590-604. 3. Kapli T, Lutteropp S, Zhang J, Kobert K, Neopilionidae, Megalopsalis, New Zealand Neopilionidae, Mangatangi, Pavlidis P, Stamatakis A, Flouri T. (2016) Bioinformatics 33(11):1630-1638. 4. Katoh K, Standley DM. (2013) Molecular Biology and Evolution 30(4):772–780. 5. Forsteropsalis, Pantopsalis, Monoscutum, Templar, Triascutum, Spinicrus. Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ. (2015) Molecular Biology and Evolution 32:268-274. 6. Miller MA, Pfeiffer W, Schwartz T. (2010) Proceedings of the Gateway Computing Environments Workshop (GCE), New Orleans, LA: 1–8.