© CSIRO 2017 Invertebrate Systematics 2017, 31, 269–301 doi:10.1071/IS16038_AC

Supplementary material

Species richness in the genus Microplana (Platyhelminthes, Tricladida, Microplaninae) in Europe: as yet no asymptote in sight

Eduardo MateosA, Ronald SluysB,D, Marta RiutortC and Marta Álvarez-PresasC

ADepartament de Biologia Evolutiva, Ecologia i Ciències Ambientals, Facultat de Biologia, Universitat de Barcelona,

Av. Diagonal 643, 08028 Barcelona, Spain.

BNaturalis Biodiversity Center, PO Box 9517, 2300 RA Leiden, The Netherlands.

CDepartament de Genètica, Microbiologia i Estadística, Facultat de Biologia and Institut de Recerca de la Biodiversitat

(IRBio), Universitat de Barcelona, Av. Diagonal 643, 08028 Barcelona, Spain.

DCorresponding author. Email: Ronald.Sluys@naturalis.nl

Table S1. List of specimens used in the molecular and histological analyses, locality of origin and codes used in the trees, Museum code for histological sections, and accession numbers for the gene sequences

Species Collecting locality Molecular code Museum code GenBank

Cox1 18S 28S EF Microplana cf. aixandrei-1 Lleida, Spain 063E024 KU867073* 252E023 KU867074* Cádiz, Spain 123D042 KU867075* 125D042 FJ969973 126D042 FJ969974 127D042 FJ969975 135D046 FJ969976a FJ970000 KU872614* - 142D042 FJ969977a 143D042 FJ969978a 146D042 KU867076* 181D055 KU867077*a Málaga, Spain 288M184 KU867078*a KU872571* KU872615* KU872658* 289M184 KU867079* 290M184 KU867080* 291M184 KU867081* 292M184 KU867082* 293M184 KU867083* 294M184 KU867084* 296M184 V.Pl. 7239.3 KU867085* Microplana cf. aixandrei-2 Lleida, Spain 061E023 KU867086* 300E023 KU867087* Barcelona, Spain 064B025 KU867088* 176B009 KU867089* 254B005 KU867090* 255B005 KU867091* 257B005 KU867092* a 302B168 KU867093* a KU872572* KU872616* KU872659* Logroño, Spain 159R050 KU867094* Girona, Spain 228G006 KU867095* 230G006 KU867096* 392G246 KU867097* a KU872573* - KU872660* 396G006 KU867098*

Cádiz, Spain 124D042 KU867099* a KU872574* KU872617* KU872661* 284D183 KU867100* 285D183 KU867101* 287D183 KU867102*

Málaga, Spain 295M184 V.Pl. 7239.1 KU867103* a KU872575* - - 297M184b V.Pl. 7239.2 -

Huesca, Spain 305H200 KU867104* 756H324 KU867105* 785H339 KU867106* 821H357 KU867107* a KU872576* KU872618* KU872662* 1000H325 KU867108* a KU872577* - KU872663*1002H325 KU867109* 1011H340 KU867110* a 1230H325 KU867111* 1231H325 KU867112* 1232H325 KU867113* 1241H476 KU867114*

Cumbria, United Kingdom 531Y269 KU867115* 532Y269 KU867116* 588Y273 KU867117* a

Saint Laurent du Pont, France 696X299 KU867118* Abruzzo, Italy 912I442 KU867119*a KU872578* - KU872664*

915I444 KU867120* a 917I446 KU867121* a 918I446 KU867122* a 919I446 KU867123* a

Viterbo, Italy 927I448 KU867124* a 928I448 KU867125* a 929I448 KU867126* a 943I451 KU867127* a KU872579* - KU872665*

Bulgaria 1224U472 KU867129* 1226U472 KU867130* 1227U472 KU867131* 1228U472 KU867132*a

Microplana astricta Sluys, Álvarez-Presas & Mateos sp. nov.

Bulgaria 1223U471 V.Pl. 7228.1 KU867128* a KU872580* KU872619* KU872666*

Microplana cephalofusca Sluys, Álvarez-Presas & Mateos sp. nov.

Bulgaria 1229U472 V.Pl. 7229.1 KU867133* a KU872581* KU872620* KU872667*

Microplana cingulata Álvarez- Bulgaria 1173U465 KU867134* a Presas, Sluys & Mateos sp. nov. 1174U465 V.Pl. 7230.1 KU867135* a KU872582* KU872621* KU872668* 1175U465 KU867136* a KU872583* KU872622* KU872669* 1176U465 KU867137* a 1177U465 KU867138* a 1181U466 KU867139* Microplana fuscomaculosa Álvarez- Oxfordshire, United Kingdom 493Y264 KU867140* Presas, Sluys & Mateos sp. nov. 495Y264 V.Pl. 7233.1 KU867141* Monmouthshire, United Kingdom 507Y267 KU867142* Cumbria, United Kingdom 546Y270 KU867143* 547Y270 KU867144* a KU872584* KU872623* KU872670* 551Y270 KU867145* Nottinghamshire, United Kingdom 591Y274 KU867146* Saint Laurent du Pont, France 692X298 KU867147* a 694X298 KU867148* a Le Chipal, France 718X304 KU867149* 725X304 KU867150* a KU872585* - KU872671* 726X304 KU867151* a KU872586* - KU872672* 727X304 KU867152* Huesca, Spain 778H335 V.Pl. 7231.1 KU867153* a KU872587* KU872624* KU872673* 780H336 V.Pl. 7231.2 KU867154* a KU872588* KU872625* KU872674* 784H338 V.Pl. 7232.1 KU867155* a 786H339 KU867156* a 801H346 KU867157* a 804H348 KU867158* 954H348 KU867159* 1018H332 KU867160* a 1050H336 KU867161* 1248H348 KU867162* 1259H325 KU867163* Microplana groga Barcelona, Spain 058B014 FJ969963a FJ969991 - - 095B037 FJ969964a FJ969992 KJ599721 KJ599693 097B038 FJ969965a FJ969993 - - Girona, Spain 390G246 KR906546a KR906664 KU872626* KU872675* Microplana henrici Bulgaria 1178U465 V.Pl. 7240.1 KU867164* a KU872589* KU872627* KU872676*

1220U471 KU867165* a KU872590* KU872628* KU872677* Microplana hyalina Barcelona, Spain 054B005 KU867166* 171B036 FJ969969a FJ969997 KU872629* KU872678* 172B036 FJ969970a 173B036 FJ969971a Saint Laurent du Pont, France 697X299 KU867167* a KU872591* KU872630* KU872679* 698X299 KU867168* a KU872592* KU872631* KU872680* 701X299 KU867169* Abruzzo, Italy 920I446 V.Pl. 7242.1 - Viterbo, Italy 930I448 KU867170* 931I448 KU867171* 932I448 V.Pl. 7241.1 - 933I449 KU867172* a 934I449 KU867173* a 936I449 KU867174* a KU872593* - KU872681* 938I449 V. Pl. 7244.1 - 942I451 KU867175* a 944I451 KU867176* a 946I451 KU867177* a - - KU872682* 947I451 V.Pl. 7243.1 - 948I452 KU867178* a Microplana kwiskea Porz-Ensen, Germany 1396Q000 V.Pl. 7245.1 - Microplana lutulenta Álvarez- Bulgaria 1172U465 KU867179* a Presas, Sluys & Mateos sp. nov. 1179U466 V.Pl. 7235.1 KU867180* a KU872594* - KU872683* 1221U471 KU867181* a KU872595* KU872632* KU872684* 1222U471 V.Pl. 7234.1 KU867182* a Microplana monacensis Navarra, Spain 204N021 KU867183* a 208N021 KU867184* KU872596* KU872633* KU872685* 738N021 V.Pl. 7246.1 KU867185* a KU872597* KU872634* KU872686* Microplana nana Girona, Spain 108G017 FJ969947a FJ969981 KJ599722 - 377G244 KR906586a KR906659 KU872635* KU872687* Huesca, Spain 308H202 V.Pl. 7213.1 KR906597a KR906657 KU872636* KU872688* 802H347 KR906600a KR906662 KU872637* KU872689* 1233H325 KR906604a Microplana nervosa Sluys, Mateos Asturias, Spain 324A219 V.Pl. 7236.2 KU867186* a KU872598* KU872638* KU872690* & Álvarez-Presas sp. nov. 325A219 KU867187* a KU872599* KU872639* KU872691*

326A220 V.Pl. 7236.1 KU867188* a 328A220 KU867189* a 329A220 KU867190* a 868A371 KU867191* a Cantabria, Spain 1132S376 V.Pl. 7237.1 KU867192* a KU872600* KU872640* KU872692* 1133S376 KU867193* a 1134S376 KU867194* a 1142S373 KU867195* KU872601* - KU872693* 1145S374 KU867196*a Microplana scharffi North Yorkshire, United Kingdom MS3 KR906625a KR906677 KU872641* KU872694* Cumbria, United Kingdom 514Y268 KR906627a 517Y268 KR906628 543Y269 KR906631a KR906678 KU872642* KU872695* Abruzzo, Italy 914I443 V.Pl. 7220.1 KR906632a Bulgaria 1182U466 KR906633a 1183U466 KR906634 Microplana sp. 1 (UCS) Saint Laurent du Pont, France 699X299 KU867197* a KU872602* KU872643* KU872696* Asturias, Spain 826A359 KU867198* a KU872603* KU872644* - Microplana sp. 2 (UCS) Bulgaria 1211U470 KU867199* a KU872604* KU872645* KU872697* 1212U470 KU867200* a KU872605* KU872646* KU872698* 1214U470 KU867201* a 1215U470 KU867202* a 1216U470 KU867203* 1217U470 KU867204* 1218U470 KU867205* a Microplana sp. 3 (UCS) Cumbria, United Kingdom 535Y269 KU867206*a 536Y269 KU867207* a 537Y269 KU867208* a 538Y269 KU867209* a - KU872647* KU872699* Microplana sp. 4 (UCS) Cumbria, United Kingdom 516Y268 KU867210* a KU872606* KU872648* - 567Y271 KU867211* a Derbyshire, United Kingdom 587Y272 KU867212* a Microplana sp. 5 (UCS) Asturias, Spain 321A218 KU867213* a KU872607* KU872649* KU872700* Microplana sparsa Sluys, Mateos & Gerês, Portugal 346Z234 KU867214* a KU872608* - KU872701* Álvarez-Presas sp. nov. 349Z234 KU867215* a 351Z234 V.Pl. 7238.1 KU867216* a

353Z235 KU867217* a KU872609* KU872650* KU872702* 354Z235 KU867218* a 355Z235 KU867219* a

Microplana terrestris Barcelona, Spain 003B002 JN379967a Navarra, Spain 086N034 FJ969951a KU872610* KU872651* -

209N021 JN379972a 218N062 FJ969979a FJ970001 KU872652* -

Huesca, Spain 303H200 JN379991 306H201 JN379993a

Asturias, Spain 322A218 JN380003a Girona, Spain 398G006 KU867220*

316G215 V.Pl. 7224.1 JN380001a KU872611* KU872653* KU872703* Buckinghamshire, United Kingdom 462Y262 KR906645a KR906682 KU872654* KU872704* Oxfordshire, United Kingdom 494Y264 KU867221* Cumbria, United Kingdom 519Y268 KU867222* Szczecin, Poland 743W309 KU867223*

745W309 V.Pl. 7200.1 KR906654 Serrahn, Germany 749Q310 KR906655 A Coruña, Spain 151C015 FJ969952a FJ969983 KJ599724 KU872705*

753C256 KU867224* a North Yorkshire, United Kingdom MT3 JN380018a KR906681 KU872655* KU872706* La Châtelaine, France 713X301 KU867225* a KU872612* KU872656* KU872707* Auliac Dessús, France 737X306 KU867226* a KU872613* KU872657* KU872708*

Unassigned specimens Cagnes sur Mer, France JL1551 KU867227 a 700X299 KU867228* a

Cádiz, Spain 179D054 KU867229* a Asturias, Spain 885A379 KU867230* a Abruzzo, Italy 916I445 KU867231* a Bulgaria 1225U472 KU867232* a

Outgroup Bipalium adventitium Kingston, USA B. adventitium AF178306 DQ666000 DQ665956 KJ599681 B. multilineatum Naka, Nagaoka, Niigata, Japan B. multilineatum HM346600 DQ666012 DQ665957 KU872709

1 Sequence obtained by Jean-Lou Justine a Cox1GMYC Dataset b For this individual there is no molecular information and hence in fact it can not be assigned to either Microplana cf. aixandrei 1 or 2 * Sequences from this study

S1 Fig. S1. Maximum likelihood tree inferred with the Cox1 barcoding dataset. Values at nodes correspond to bootstrap and posterior probability values.

Fig. S2. Maximum likelihood trees inferred with the information of individual nuclear genes and with concatenation of sequences belonging to the three genes: (A) 28S rDNA, (B) elongation factor 1-α, (C) 18S rDNA and (D) nuclear concatenated dataset. Asterisks at nodes correspond to bootstrap and posterior probability values greater than 75% and 0.85, respectively. In D, red asterisks correspond to nodes supported only in the Bayesian inference analysis.

Figs S3–S6. Microplana astricta. Holotype, ZMA V.Pl. 7228.1. S3, Ventral subepidermal and parenchymal musculature, sagittal section; S4, sagittal section through the copulatory apparatus; S5, sagittal section through male atrium and penis papilla; S6, sagittal section of female copulatory apparatus, showing muscular constriction in bursal canal.

S3 S4 200 µm

lpm

cb

pbvnc

ppbc

lpm vd

50 µm go

S5 cb

S6cb

pp bc pp

100 µm go go 100 µm

S7 S8

ph pp

te

200 µm 100 µm

Figs S7–S11. Microplana cephalofusca and M. cingulata. S7, Microplana cephalofusca, holotype ZMA V.Pl. 7229.1, sagittal section, showing testes follicles. S8, Microplana cephalofusca, holotype ZMA V.Pl. 7229.1, sagittal section through penis papilla. S9, Microplana cephalofusca, holotype ZMA V.Pl. 7229.1, sagittal section through copulatory bursa. S10, Microplana cingulata, holotype ZMA V.Pl. 7230.1, sagittal section through pharynx, showing the various muscle layers. S11, Microplana cingulata, holotype ZMA V.Pl. 7230.1, sagittal section of copulatory apparatus, showing the penis papilla, tube-shaped fold and copulatory bursa.

S9 inS10

cb

pp

bc

100 µm lu

S11

ppcb

50 µm

tsf pp 200 µm

S13

S12 50 µm

te vep 100 µm

S14 pb

lu

fgd

100 µm pp go

S15 in

S16

pe gs

fgd

od

50 µm 100 µm vep

Figs S12–S16. Microplana fuscomaculosa. S12, Paratype, ZMA V.Pl. 7231.2, sagittal section through pharynx. S13, ZMA V.Pl. 7232.1, sagittal section, showing the testes. S14, Paratype, ZMA V.Pl. 7231.2, sagittal section through the penis,showing the broad zone of circular muscle around bulbar and penial lumen. S15, Holotype, ZMA V.Pl. 7231.1, sagittalsection of communication between female genital duct and gut via an intestinal appendix. S16, ZMA V.Pl. 7232.1, sagittalsection showing opening of female genital duct into the globular sac.

S17 S18

gid in bc pp

cb pp

100 µm

vep 100 µm

Figs S17–S21. Microplana lutulenta, M. nervosa and M. sparsa. S17, Microplana lutulenta, holotype, ZMA V.Pl. 7234.1, sagittal section through penis and female genital duct. S18, Paratype, ZMA V.Pl. 7236.2, sagittal section of copulatory apparatus. S19, Microplana nervosa, holotype, ZMA V.Pl. 7236.1, sagittal section, showing communication between copulatory bursa and gut, as well as muscularised intestinal extension. S20, Microplana nervosa, holotype, ZMA V.Pl. 7236.1, sagittal section of penis and muscularised intestinal extension. S21, Microplana sparsa, holotype, ZMA V.Pl. 7238.1, sagittal section of the copulatory apparatus.

S19 S20 100 µm

inin

ie pp

cbie

vd

100 µm

S21

sg

fgd pp

100 µm

S22 S23

rh spt cb

50 µm

ch vep

50 µm

Figs S22–S26. Microplana cf. aixandrei and M. henrici. S22, Microplana cf. aixandrei, ZMA V.Pl. 7239.2, sagittal section of bursa, containing remnants of spermatophore. S23, Microplana cf. aixandrei, ZMA V.Pl. 7239.2, sagittal section of anterior ventral epidermis, showing rhamnites and chondrocysts. S24, Microplana henrici, ZMA V.Pl. 7240.1, sagittal section, showing the testes. S25, Microplana henrici, ZMA V.Pl. 7240.1, sagittal section of copulatory apparatus, showing the annular fold. S26, Microplana henrici, ZMA V.Pl. 7240.1, sagittal section of male copulatory apparatus.

S24 S25bl

in

fgd

af

te 100 µm

S26

te

bl af pp

vep 100 µm af

100 µm

S28 S27

ov pp

am af od

af af

200 µm 50 µm vep

Figs S27–S29. Microplana peneckei and M. hyalina. S27, Microplana peneckei, holotype, NHMW, Inv. 2851, sagittal section of penis papilla and atrium, showing three annular folds. S28, Microplana hyalina, ZMA V.Pl. 7241.1, sagittal section, showing ovary, ampulla and oviduct. S29, Microplana hyalina, ZMA V.Pl. 7242.1, sagittal section of copulatory bursa, showing coloured balls in vacuoles of lining epithelium.

S29

cb

50 µm