Mind the outgroup: influence of taxon sampling on total-evidence dating of pimpliform parasitoid (, )

TAMARA SPASOJEVIC, GAVIN R. BROAD, ILARI E. SÄÄKSJÄRVI, MARTIN SCHWARZ, MASATO ITO, RIKIO MATSUMOTO, STANISLAV KORENKO AND SERAINA KLOPFSTEIN

Basel, 25 June 2018

220 200 180 160 140 120 100 80 60 40 20 0 Pimpliformes morphology (Wahl and Gauld 1998) + 28S (Quicke et al. 2009, 2015) Coleoptera Diptera

? Symphyta

Ichneumonidae

Araneae

Lepidoptera

Siricidae

Endoparasitoids Ectoparasitoids

Idiobionts Koinobionts Pimpliformes morphology (Wahl and Gauld 1998) + 28S (Quicke et al. 2009, 2015) Coleoptera Diptera

? Symphyta Aculeata

Ichneumonidae

Araneae

Lepidoptera

Siricidae

Endoparasitoids Ectoparasitoids ? Idiobionts 60 40 20 0 Koinobionts 120 100 80 60 40 20 0 Establishing evolutionary timescale

taxon 1

taxon 2

taxon 3 Establishing evolutionary timescale

taxon 1

taxon 2

taxon 3

≥ 50 Ma

50 Ma Node dating taxon 1 - only the oldest reliable fossil taxon 2

taxon 3 - how to model node age probability? (Warnock et al. 2011, 2015)

0 Million years ago Establishing evolutionary timescale

taxon 1

taxon 2

taxon 3

50 Ma ≥ 50 Ma ?

50 Ma Node dating taxon 1 - only the oldest reliable fossil taxon 2

taxon 3 - how to model node age probability? (Warnock et al. 2011, 2015)

0 Million years ago Establishing evolutionary timescale

taxon 1

taxon 2

taxon 3

50 Ma Total evidence dating taxon 1 (Ronquist, Klopfstein et al. 2012)

taxon 2 - all available fossils

taxon 3 - no need for prior taxonomic placement

million years ago 0 Total evidence dating - TED

Bayesian analysis 0

1 Total evidence dating - TED

Bayesian analysis 0

1

Can we reconstruct a stable phylogeny of Pimpliformes?

How accurately and precisely can fossils be placed? TED dataset Sanger sequencing: 11 genes

28S, CAD, COI 8 genes TED dataset Sanger sequencing: 11 genes

10 genes 4011 base pairs 28S, CAD, COI 8 genes 1st and 2nd codon positions only TED dataset 222 adult morphological characters (Gauld et. al 2002, Bennett in prep.)

> 56 000 cells binary: 34% multistate: 66% continuous: 13 characters new or modified: 43%

completness of coding ntaxa min. max. mean extant 151 31% 99% 80% fossil 79 5% 54% 22% TED dataset TED dataset Sanger sequencing: 11 genes

10 genes 4011 base pairs 28S, CAD, COI 8 genes 1st and 2nd codon 211 extant taxa positions only 79 fossils

222 adult morphological characters (Gauld71% et. aloverlap 2002, Bennett in prep.)

> 56 000 cells binary: 34% multistate: 66% continuous: 13 characters new or modified: 43%

completness of coding ntaxa min. max. mean extant 151 31% 99% 80% fossil 79 5% 54% 22% Morphology only Bayesian non-clock analysis

Ephialtini

Theroniini

Delomeristini

Pimpliformes Ichneumonidae

Pimplini

Majority-rule consensus tree without fossils Spasojevic et al. (in prep.) Bayesian non-clock analysis

Good resolution at the backbone Ephialtini of Pimpliformes (pp > 0.95)

Theroniini Delomeristini

Five of the nine subfamilies Pimpliformes

Ichneumonidae monophyletic

Acaenitinae and Orthocentrinae supported

Pimplini

Long outgroup branches

Majority-rule consensus tree without fossils Spasojevic et al. (in prep.) Pimplinae

Option 1: Pimplinae and Ephialtinae ? Option 2: Xanthopimplinae and Pimplinae Option 3: Pimplinae Pseudopimpla (Hebermehl, 1917)

? Pimplinae Acaenitinae

Choi et al. 2018 Diptera parasitoids

Orthocentrinae + Diacritinae ? + = Orthocentrinae Cylloceriinae TED analysis

Bayesian inference, MrBayes (Ronquist et al. 2012)

Molecular data: IGR clock model PartitionFinder2, <30 parsimony informative sites Linked clocks GTR+G+I Uniform tree prior Topological constraints Morphological data: for rooting Mkv model (Levis 2001; Puttick et al. 2017) TED analysis

Bayesian inference, MrBayes (Ronquist et al. 2012)

Molecular data: IGR clock model PartitionFinder2, <30 parsimony informative sites Linked clocks GTR+G+I Uniform tree prior Topological constraints Morphological data: for rooting Mkv model (Levis 2001; Puttick et al. 2017)

Klopfstein and Spasojevic (2019) PLOS ONE TED analysis

Bayesian inference, MrBayes (Ronquist et al. 2012)

Molecular data: IGR clock model PartitionFinder2, <30 parsimony informative sites Linked clocks GTR+G+I Uniform tree prior Topological constraints Morphological data: for rooting Mkv model (Levis 2001; Puttick et al. 2017)

Klopfstein and Spasojevic (2019) PLOS ONE TED analysis

Bayesian inference, MrBayes (Ronquist et al. 2012)

Molecular data: IGR clock model PartitionFinder2, <30 parsimony informative sites Linked clocks GTR+G+I Uniform tree prior Topological constraints Morphological data: for rooting Mkv model (Levis 2001; Puttick et al. 2017)

Klopfstein and Spasojevic (2019) PLOS ONE TED analysis

Evanioidea

Ichneumonidae Pimpliformes

225 200 175 150 125 100 75 50 25 0 Ma Posterior probability 0.6 1 TED analysis

Evanioidea

Ichneumonidae Pimpliformes

225 200 175 150 125 100 75 50 25 0 Ma Posterior probability The oldest Hymenoptera fossil 0.6 1 TED analysis

Evanioidea

Did the fossil placement influence the age estimates?

Did the outgroup choice influence the age estimates?

Ichneumonidae Pimpliformes

225 200 175 150 125 100 75 50 25 0 Ma Posterior probability The oldest Hymenoptera fossil 0.6 1 Where were the fossils placed? Where were the fossils placed?

Most of the fossils had similar placement

Highest probabilty of placement on branches to extant genera (Banchinae)

… and on morphologically empty branches Outgroup setting

Evanioidea full outgroup

Ichneumonidae Pimpliformes Outgroup setting

Evanioidea full outgroup

Braconidae (7)

Ichneumonidae Pimpliformes Outgroup setting

Evanioidea full outgroup

Braconidae (7)

one braconid

Ichneumonidae Pimpliformes Outgroup setting

Evanioidea full outgroup Tanychorinae Braconidae (7)

one braconid

Tanychorinae

Ichneumonidae Pimpliformes Outgroup setting

Evanioidea full outgroup

Braconidae (7)

one braconid

Tanychorinae

Xoridinae

Ichneumonidae Pimpliformes TED analysis – age estimates

Median age estimates in million years

300

250

(7)

Jurassic s 200 Cretaceou (1)

100 Full outgroup Braconidae Tanychorinae Xoridinae TED analysis – age estimates

Median age estimates in million years

300 Permian

250 Triassic

(7)

Jurassic s 200 Cretaceou (1)

100 Full outgroup Braconidae Tanychorinae Xoridinae TED analysis – age estimates

Median age estimates in million years

300 Permian

250 Triassic

(7)

Jurassic s 200 Cretaceou (1)

100 Full outgroup Braconidae Tanychorinae Xoridinae

Placement of the Cretaceous fossils is an artefact

Distant outgroups: long branches, no fossils and almost no morphological data When did the Pimpliformes originate and diversify?

̴ 23 Ma

̴ 66 Ma

̴ 100 Ma

̴ 145 Ma

̴ 201 Ma When did the Pimpliformes originate and diversify?

̴ 23 Ma

The oldest fossil of Pimpliformes

̴ 66 Ma The oldest fossil of an extant subfamily?

̴ 100 Ma

The oldest fossil of Ichneumonidae ̴ 145 Ma

̴ 201 Ma When did the Pimpliformes originate and diversify?

Ichneumonidae

̴ 23 Ma

The oldest fossil of Pimpliformes

̴ 66 Ma The oldest fossil of an extant subfamily?

̴ 100 Ma Peters et al. 2017

The oldest fossil of Ichneumonidae ̴ 145 Ma

Our estimate

̴ 201 Ma When did the Pimpliformes originate and diversify?

Pimpliformes

̴ 23 Ma

The oldest fossil of Pimpliformes

̴ 66 Ma Peters et al. 2017 The oldest fossil of an extant subfamily?

̴ 100 Ma

The oldest fossil of Ichneumonidae ̴ 145 Ma

Our estimate

̴ 201 Ma When did the Pimpliformes originate and diversify?

Pimpliformes

̴ 23 Ma

The oldest fossil of Pimpliformes

̴ 66 Ma Peters et al. 2017 The oldest fossil of an extant subfamily?

̴ 100 Ma

The oldest fossil of Ichneumonidae ̴ 145 Ma (Misof et al. 2014)

Our estimate

̴ 201 Ma (Tong et al. 2015) What do we need to date Pimpliformes and Ichneumonidae?

Thorough examination and even sampling of fossils

Thorough sampling of extant taxa - especially outgroup - eliminate morphology empty branches

New molecular datasets ??? Acknowledgments

Andrew Bennett Sonja Wedmann Dmitry Kopylov Thalia Karim David Zelagin Marsh Finnegan Alan M. Rulis Richardo Pérez-de la Fuente Bernhard Hostettler Ursula Menkveld-Gfeller Achim Reisdorf Christian Kropf