Lousy Phylogenies: Phthiraptera systematics and the antiquity of lice

Vincent S. Smith

Division of Environmental and Evolutionary Biology Institute of Biomedical and Life Sciences Graham Kerr Building University of Glasgow Scotland, UK Lice (Phthiraptera): biology i. Diversity • 4,384 spp “chewing lice” (3,508 bird & 402 mammal spp) • 543 spp sucking lice (812 spp mammal) ii. Physical characteristics • wingless, dorsoventrally flattened, 0.3-11mm, 3 nymphal instars • feed on feathers, fur & skin debris (“chewing lice”) or blood • adaptations against host grooming iii. Distribution • mirror host distribution worldwide in virtually every habitat • absent on Chiroptera, Cetacea, Microbiotheria, Monotremata, Notoryctemorphia, Pholidota & Sirenia. iv. Cophylogeny, cospeciation & coevolution Lice (Phthiraptera): taxonomy Chewing Lice Sucking Lice “Mallophaga” (Anoplura)

Amblycera Ischnocera Rhynchophthirina Anoplura

- 1,350 spp in 6 families - 3,060 spp., # families ? - Just 3 species - 550 spp. in 16 families - Most “primitive” - Widespread, ~90% spp. - On unrelated hosts - Absent from birds suborder on birds - Acquired via shared use - Well studied - More vagile, less host - Avian groups poorly of watering holes? - Greatest medical & specific known - Tiny mandibles present veterinary significance - Widespread on birds - Highly host specific at end of a long rostrum - Vectors of several - 2 fam. South American - Niche restricted pierce hosts thick skin important diseases mammals & marsupials - Mammal infesting - Low prevalence on - 1 fam. on Australasian species of veterinary healthy hosts marsupials significance Lice (Phthiraptera): phylogeny ChewingParasitic Lice Lice Sucking Lice “Mallophaga(Phthiraptera” ) (Anoplura)

Psocoptera Amblycera Ischnocera Rhynchophthirina Anoplura

Lyal, 1985 (Morphology) Kim & Ludwig, 1982 (Morphology) Suborder Family Genus

Anoplura 532 AnopluraPhthiraptera: Rhyn… 3 RhynchophthirinaPhylogenetic resolution, 1996

3,060 Ischnocera

Ischnocera

1,344 AmblyceraBirds Amblycera Mammals Suborder Family Genus

Anoplura 532 Phthiraptera: Rhyn… 3 Phylogenetic resolution, 2003

3,060

Ischnocera

1,350 Birds Amblycera Mammals 1996 2003 Suborder Family Genus Suborder Family Genus

Anoplura 532 541

Rhyn… 3 3

3,060 3,060

Ischnocera

1,344 1,350

Amblycera Amblyceran Families

Menoponidae

Boopiidae 85 Birds Worldwide & Australasian Marsupials Laemobothriidae

Ricinidae

Trimenoponidae

Protogyropinae

84 Gyropidae South/Central American Gyropinae Rodents & Marsupials Analysed by Marshall, 2003 Grilicolinae from Clay, 1970 (Morphology) Liposcelidae (Psocoptera) Avian Amblycera

Liposcelis bostrychophilus 100 Trochiloecetes rupununi Ricinidae 100 93 12 85 Ricinus fringillae - Hummingbirds 3 Trochiliphagus abdominalis Laemobothrion maximum L Laemobothriidae 100 Therodoxus oweni 100 98 100 Boopia tarsata - Falconiforms 8 61 75 Boopidae 15 Paraheterodoxus insignis B 1 61 100 96 Latumcephalum lesouefi/macropus 88 3 99 - Australasian marsupials 9 Paraboopia flava 7 Rediella mirabilis 89 76 Actornithophilus uniseriatus 4 90 Plegadiphilus threskiornis 74 Chapinia robusta 5 Bonomiella columbae 1 62 Pseudomenopon pilosum Dennyus-complex 2 2 Ancistrona vagelli 98 96 Dennyus hirundinis - Birds worldwide 3 Myrsidea victrix A 1 Holomenopon brevithoracicum Eidmanniella pellucida 1 1 Machaerilaemus laticorpus/latifrons Austromenopon-complex 1 Austromenopon crocatum Neomenopon pteroclurus - Aquatic birds 1 Hohorstiella lata M B Osborniella crotophagae 1 52 Eomenopon denticulatum 3 1 Piagetiella bursaepelecani Ciconiphilus quadripustulatus 1 1 Cuculiphilus fasciatus Colpocephalum-complex 2 80 Ardeiphilus trochioxus 57 92 Odoriphila clayae/phoeniculi - Birds worldwide 4 75 1 Psittacomenopon poicephalus 5 51 87 C 1 73 Colpocephalum zebra 3 Comatomenopon elbeli/elongatum Gruimenopon longum 2 Hoazineus armiferus 1 Meromenopon meropis Trinoton anserinum 2 64 Marshall, 2003 57 Menacanthus stramineus Menacanthus/Menopon-cpx. 1 1 Colimenopon urocolius - Birds worldwide 44 genera, 147 char. 2 Somaphantus lusius Amyrsidea ventralis 2 D Menopon gallinae (Morphology) 1 2 Numidicola antennatus

First Fossil Louse (Amblycera: Menoponidae)

Psocoptera • Collected Eckfeld maar near • Parasite of Anseriformes or Ricinidae Eifel, Germany Charadriiformes • Middle EoceneLae m(Middleobothriida eLutetian, • Crown group position 44.3±0.4 Ma) • Points to a long coevolutionary Boopiidae history with birds • First fossil louse Dennyus • Suggests lice are very old • Completes ordinal- comple xrepresentation • Useful molecular calibration point of fossil insects?Austromenopon - complex • Excellent preservation Menacanthus / • PhylogeneticM affinitiesenopon - co mwithplex modern featherColp olicecephalum - complex

Wappler, Smith & Dalgleish , In prep. Ischnoceran Families Suborder Family Genus

Anoplura 532

Rhyn… 3

“PhilopteridaeGoniodidae ” Ischnocera 3,060

Ischnocera

HeptapsogasteridaeHeptapsogasteridae Trichodectidae

Smith,Hopkins 2000 & Clay, 1951 Generic level phylogeny of 1,344 GoniodidaeBirdscf. Eicher, & Heptapsogasteridae 1961 Amblycera (Morphology)21 families Mammals Avian Ischnoceran Phylogeny Haffneria grandis Harrisoniella hopkinsi Paraclisis diomedea PerineusPhiloceanus nigrolimbatus Smith, 2001 Halipeurus pelagicus Philoceanus-complex garrodiae Generic level phylogeny Pseudonirmus gurlti Naubates fuliginosus rd Pelmatocerandra setosa Approx. 1/3 of all ischnoceran genera Ardeicola smithersi Anaticola crassicornis Pectinopygus bassani 51 genera, 138 characters Pectinopygus sulae Aquanirmus australis Ibidoecus plataleae (Morphology) Trabeculus schillingi Craspedonirmus colymbinus QuadracepsSaemundssonia coenocoryphae Docophoroides brevis Saemundssonia-complex desolata Saemundssonia haematopi Podargoecus strigoides Alcedoecus delphax CraspedorrhynchusPhilopterus platystomus Strigiphilus vapidus Sturnidoecus-complex sturni Philopterus ornatus Vernoniella guimarãesi Acidoproctus rostratus Acidoproctus hilli Rallicola lugens Osculotes curtus Osculotes macropoda Coloceras damicorne Campanulotes bidentatus Multiple clades of lice GoniocotesGoniodidae gallinae Goniodes pavonis Goniodes kéleri on the same host Chelopistes guttatus Austrogoniodes waterstoni Strongylocotes angulocapitis DiscocorpusHeptap. c. cephalosus Columbicola columbae SplendoroffulaLipeurus ruwenzorornis Oxylipeurus dentatus Cuclotogaster-complex madagascariensis Lipeurus caponis Degeeriella rufa UpupicolaDegeeriella upupae Lagopoecus affinis Syrrhaptoecus-complex falcatus Neopsittaconirmus borgiolii Paragoniocotes rotundus Brüelia semiannulata Falcolipeurus affulgeus Archolipeurus nandu Geomydoecus (G.) heaneyi Felicola (F.) viverriculae TrichodectesTrichodectidae (T.) melis Bovicola limbatus Damalinia (D.) crenelata Characters Defining Major Ischnoceran Clades

A. Circumfasciate Head

B. Non-Circumfasciate Head

N I N II N III Adult

C. Wing Lice D. Body Lice E. Head Lice Characters Defining Major Ischnoceran Clades

PhiloceanusWing Problems with Morphology! -complexLice Smith et al, 2003

• Incongruent with molecularNon- data • Morphological convergencecircumfasciate • Molecular data suggests many of SaemundssoniaHead these pairs are more closely related Lice-complex • Character displacement? Philopterus • Sympatric speciation? -complex

Body Circumfasciate GoniodidaeLice

CI: 1.0 Heptap. RI: 1.0 LipeurusWing -complex Lice Degeeriella -complex Multiple clades occupy different CI: 0.5 host niches TrichodectidaeRI: 0.95 Rhynchophthirina: Haematomyzidae

Suborder Family Genus

Anoplura 532 3 Rhynchophthirina

3,060

Ischnocera

1,344

Amblycera Rhynchophthirina: Haematomyzidae

• Just 3 species in 1 family • Present on elephants, wart hog & bush pig (perhaps also mammoths?) • Monophyly & sister placement with Anoplura well supported • Chewing lice (have outwardly opposing mandibles) • Pool blood feeders (telmophages) cf vessel feeding Anoplura • Spread via shared use of watering holes? Haematomyzus elephantis • Possible cryptic species? ex Asian / African elephant (morphologically identical species on African & Asian elephants) • Specimens hard to come by! Haematomyzus elephantis Haematomyzus hopkinsi Haematomyzus porci Part of Smith, in prep. 51 genera, 76 species 184 characters (Morphology) Anopluran Families

Anoplura At least 15 532 distinctAnoplura families Rhyn… 3

3,060

Ischnocera

1,344

Amblycera Anoplura: familial phylogeny

• 541spp. in 15 families • Patchily distributed across mammals

• Monophyly of all 15 families supported • 3 basal clades • Many lice of host groups (including Primates) paraphyletic

Based on Kim, 1988 47 genera, 39 characters (Morphology) Kim’s dataset OUTGROUP “Polyplacidae” 1 re-analysed: “Polyplacidae” 2 “Polyplacidae” 3 Hoplopleuridae • Length=219 (CI=0.26, RI=0.62) “Polyplacidae” 4-12 • Paraphyly of 2 of the 5 families Echinophthiriidae that were not monogenic Enderleinellidae • Just 1 node agrees with Kim’s “Linognathidae” 1 original tree! Neolinognathidae • Only 3 other nodes are “Linognathidae” 2 compatible with Kim’s tree Pedicinidae Pthiridae “Polyplacidae” 13 Ratemiidae Hamophthiriidae Hybophthiridae Pediculidae Pecaroecidae Haematopinidae Kim, 1988 reanalysed Microthoraciidae Strict Consensus Haematomyzus elephantis HaematomyzusHaematomyzidae hopkinsi Haematomyzus porci MicrothoraciusMicrothoraciidae cameli Ratemia squamulata Ratemiidae HamophthiriusHamophthiriidae galeopitheci Hybophthirus notophallus Anoplura: Haematopinus asini Hybophthiridae HaematopinusHaematopinidae suis Pecaroecus javalii Pedicinus ferrisi Pecaroecidae Pedicinus patasPedicinidae phylogeny Pthirus gorillae Pthirus pubisPthiridae Pediculus schaeffi Pediculus humanus Pediculus mjobergiPediculidae Pediculus humanus var. americanus Scipio aulacodi Scipio tripedatus“Polyplacidae” 1 Neolinognathus sp. NeolinognathusNeolinognathidae elephantuli Solenopotes muntiacus Prolinognathus aethiopicus Prolinognathus ferrisi LinognathusLinognathidae setosus Linognathus aepycerus Linognathus africanus Latagophthirus rauschi Echinophthirius horridus Lepidophthirus macrorhini Antarctophthirus callorhini AntarctophthirusEchinophthiriidae microchir Proechinophthirus fluctus Proechinophthirus zumpti Johnsonpthirus chlorotalpae Johnsonpthirus heliosciuri Lemurphthirus galagus Lemurphthirus stigmosus Linognathoides laeviusculus Sathrax durus Neohaematopinus callosciuri Neohaematopinus inornatus • Why are rodents so lousy? Abrocomaphthirus chilensis Ctenophthirus cercomydis Proenderleinellus calva Polyplax abyssinica“Polyplacidae” 2 • Why are other mammals Polyplax spinulosa Docophthirus acinetus Eulinognathus denticulatus relatively louse free cf. birds Haemodipsus leporis Haemodipsus lyriocephalus Lagidiophthirus parvus Fahrenholzia ferrisi • Good evidence for Fahrenholzia pinnata Galeophthirus caviae Phthirpediculus brygooi cophylogeny in some clades Phthirunculus sumatranus Atopophthirus emersoni Microphthirus uncinatus EnderleinellusEnderleinellidae osborni Werneckia nigeriensis Hoplopleura acanthopus Paradoxophthirus emarginata • Comparative studies underway Smith, in prep. Hoplopleura pacifica HoplopleuraHoplopleuridae irritans using these trees 51 genera, 76 species Pterophthirus audax Pterophthirus splendida Ancistroplax crocidurae 184 characters Haematopinoides squamosus Typhlomyophthirus“Polyplacidae bifoliatus ” 3 (Morphology) Schizophthirus graphiuri Schizophthirus pleurophaeus Louse Molecular Studies

Mitochondrial Nuclear

- COI (400-1500bp) - EF1-α (350-450bp) - 12S rRNA (450bp) - 18S rRNA (1500+bp) - Cyt. B (450bp) - Wingless, PEPCK, DDC - ND4 (500bp)

• Easy to amplify • Hard to amplify • Limited phylogenetic utility • Multiple copies outside families • RNA alignment issues • Often restricted to use within • Utility varies between genes & closely related genera taxa

Need New Genes! Relative Substitution Rate (Mit. COI vs nuc. EF1-α)

Rate Parameter Data Sites Ratio Set COI EF1-α (COI:EF1α) all sites 1 0.018 55.6 pos. 1 1 0.013 76.9 Physconelloidinae pos. 2 0.12 0.016 7.5 Lice pos. 3 129.9 0.41 316.8 all sites 1 0.016 61.5 pos. 1 1 0.039 25.6 Columbicola pos. 2 0.15 0.017 8.8 pos. 3 169.78 0.8 212.2 all sites 1 0.25 4.1 pos. 1 1 0.20 5.0 aphids pos. 2 0.0001 0.0001 1.0 pos. 3 44.79 2.49 18.0 Austrophilopterus Austrogoniodes cristati subsimilis Louse 12S Austrophilopterus Naubates prioni subsimilis Secondary Campanulotes bidentatus Structure

Oxylipeurus chiniri

Halipeurus pelagicus Austrogoniodes waterstoni

Austrogoniodes cristati

Naubates harrisoni

mit. 12S rRNA Columbicola columbae Rallicola columbiana Page et al, 2002 Segment 1: 33-33' Segment 2: 36-38 160 160 140 140 120 120 100 100 80 insects 80 60 60 40 40 20 20 0 0 12S Length Length Length 7 12 6 10 Variation 5 lice 8 4 6 3 4 2 2 1

0 0 210220230240250260270280290300310320 5 10 15 20 25 30 35 40 45 Length Length

200 Segment 3: 38'-39' 120 Segment 5: 34'-33' 180 100 160 140 80 120 100 60 80 40 60 40 20 20 0 0 Length Length 14 10 9 12 8 10 7 8 6 5 6 4 Insects vs. 4 3 2 2 Lice 1 0 0 15 20 2530 35 40 45 50 55 60 65 70 75 80 85 35 40 45 50 55 60 65 70 75 80 Page et al, 2002 Length Length Louse Mitochondrial Genome

Gene arrangement of Heterodoxus macropus (Amblycera)

CMWERII S2 L2 GL1 KNAY DIFS1 QV PT

cox1 cox3 nad1 cob nad4 nad2 nad5 cox2 rrnL rrnS atp6 atp8 nad3 nad6 nad4L

cox1 cox2 cox3 nad5 nad4 cob nad2 rrnL rrnS nad2 atp6 atp8 nad3 nad6 nad4L

L KD G ARNS EF H TP S V IQM WCY 2 1 2 L1 Inferred gene arrangement of ancestral insect

Adapted from Shao, Campbell & Barker, 2001 Louse Molecular Studies

Mitochondrial Nuclear

- COI (400-1500bp) - EF1-α (350-450bp) - 12S rRNA (450bp) - 18S rRNA (1500+bp) - Cyt. B (450bp) - Wingless, PEPCK, DDC - ND4 (500bp)

• Easy to amplify • Hard to amplify • Limited phylogenetic utility • Multiple copies outside families • RNA alignment issues • Often restricted to use within • Utility varies between genes & closely related genera taxa

Need New Genes! RHYN… Louse Nuclear ANOPLURA Genes

What Not to do!

• 350 bp single copy EF1- ISCHNOCERA α • Limited phylogenetic utility above families

AMBLYCERA

Cruickshank et al, 2001 Phylogenetic Utility 18S rRNA

Smith et al, 2003 Conclusions… • Much recent progress on basal phthirapteran phylogeny • Monophyly of suborders well supported • Complex pattern of host-louse relationships • Distinctive molecular evolution in lice • Incongruence between molecules & morphology

Ongoing Questions…

• To what extent do lice cospeciate with their hosts? • Why are some hosts more lousy than others? • How old are the major louse clades? • Are Phthiraptera truly paraphyletic? Acknowledgements

Collaboration Stephen Barker Dale Clayton Tom Ford Kevin Johnson Rod Page Isabel Marshall David Reed

Funding Wellcome Trust, UK Natural Environment Research Council, UK Pairwise ML Corrected Distances for 3rd Sites (Nuclear EF1-α against Mit. COI)

300

200 Positions) rd 3 100

(COI Slope

ML Corrected Divergences approximates 0 relative rate 0 0.1 0.2 0.3 0.4 0.5 0.6 Around 1000:1 !!! ML Corrected Divergences (EF1-α 3rd Positions) Johnson et al, 2003 18S – not a panacea for basal louse Phylogeny!

Trogiomorph Psocoptera Liposcelis sp. Trogiomorph Psocoptera Amblycera Ischnocera

Liposcelis sp. Ischnocera Amblycera

Anoplura Anoplura Bayesian Uncorrected NJ

0.1 0.1