Table 1. GenBank accession numbers for COI, 16S, ITS2, and 28S DNA sequences. All sequences are new, except the sequences of the outgroups which were obtained from our previous studies (Dayrat et al. 2011, 2016, 2017, 2018, 2019; Dayrat and Goulding 2017; Goulding et al. 2018a, b, c).SpeciesIndividual (DNA #)LocalityGenBank COIGenBank 16SGenBank ITS2GenBank 28SAlionchis jailoloensis5137Halmahera, IndonesiaMG953528MG953538MG953548MK122918Marmaronchis marmoratus5409Madang, Papua New GuineaMK122838MK122859MK122893MK122915Marmaronchis vaigiensis1183SingaporeMK122812MK122854MK122877MK122910Melayonchis aileenae970Peninsular MalaysiaKX240033KX240057MK122902MK125514Melayonchis eloisae1011SingaporeKX240026KX240050MK122904MK125515Onchidella celtic a5013FranceMG958715MG958717MK122906MK122921Onchidella nigricans1524New South Wales, AustraliaMG970878MG970944MK122908MK122923Onchidina australis1523New South Wales, AustraliaKX179548KX179561MG958719MG958887Onchidium stuxbergi5605VietnamKX179520KX179537MG958721MG958886Onchidium typhae965Peninsular MalaysiaKX179509KX179525MG958720MG958885Paromoionchis daemelii1511New South Wales, AustraliaMH055048MH055129MH055241MH055289Paromoionchis tumidus1732Sumatra, IndonesiaMH054951MH055104MH055196MH055268Peronia sp.696Okinawa, JapanHQ660043HQ659911MG958871MG958883Peronia sp.706Hawaii, USAHQ660038HQ659906MG958722MG958884Peronina tenera960Peninsular MalaysiaMG958740MG958796MG958840MG958874Peronina zulfigari6005Peninsular MalaysiaMG958775MG958831MG958867MG958882Platevindex luteus1001SingaporeMG958714MG958716MG958718MG958888Wallaconchis ater5121Halmahera, IndonesiaMG970820MG970911MG971134MG971186Wallaconchis sinanui2740Ambon, IndonesiaMG970713MG970881MG971093MG971161L. boucheti1004SingaporeMH619242MH6193031005SingaporeMH619243MH619304MH619364MH6194131037Brunei DarussalamMH619244MH619305MH6193651038Brunei DarussalamMH619245MH619306MH619366MH6194141679Northern Territory, AustraliaMH619246MH6193071681Northern Territory, AustraliaMH619247MH619308MH619368MH6194161685Northern Territory, AustraliaMH619248MH6193091688 HNorthern Territory, AustraliaMH619249MH6193101729Sumatra, IndonesiaMH619250MH619311MH6193692559Queensland, AustraliaMH619251MH619312MH6193702578Queensland, AustraliaMH619252MH619313MH6193712593Queensland, AustraliaMH619253MH619314MH6193722604Queensland, AustraliaMH619254MH619315MH619373MH6194172609Queensland, AustraliaMH619255MH619316MH619374MH6194182612Queensland, AustraliaMH619256MH619317MH6193752692Queensland, AustraliaMH619257MH619318MH619376MH6194192693Queensland, AustraliaMH619258MH6193193615Bohol, PhilippinesMH619259MH619320MH619377MH619420914Peninsular MalaysiaMH619260MH619321MH619378MH619421915Peninsular MalaysiaMH619261MH619322MH6193795520Peninsular MalaysiaMH619262MH619323MH6193805609VietnamMH619263MH619324MH6193815610VietnamMH619264MH619325MH619382MH619422L. bourkei bourkei1656Northern Territory, AustraliaMH619290MH6193511616Northern Territory, AustraliaMH619291MH619352MH619402MH6194321617Northern Territory, AustraliaMH619292MH619353MH6194031618Northern Territory, AustraliaMH619293MH6193541621Northern Territory, AustraliaMH619294MH619355MH6194041652Northern Territory, AustraliaMH619295MH619356MH6194051657 HNorthern Territory, AustraliaMH619296MH619357MH6194061659Northern Territory, AustraliaMH619297MH619358MH6194071666Northern Territory, AustraliaMH619298MH619359MH6194081673Northern Territory, AustraliaMH619299MH619360MH619409MH6194341692Northern Territory, AustraliaMH619300MH619361MH6194101693Northern Territory, AustraliaMH619301MH619362MH619411MH6194351694Northern Territory, AustraliaMH619302MH619363MH619412MH619436L. bourkei lateriensis2743Ambon, IndonesiaMH619284MH619345MH619396MH6194292753Ambon, IndonesiaMH619285MH619346MH619397MH6194306061Ambon, IndonesiaMH619286MH619347MH6193986063Ambon, IndonesiaMH619287MH619348MH6193996064 HAmbon, IndonesiaMH619288MH619349MH619400MH6194316065Ambon, IndonesiaMH619289MH619350MH619401L. bourkei matangensis978SingaporeMH619265MH619326MH619383MH619423979SingaporeMH619266MH619327MH619384MH619424980SingaporeMH619267MH619328983SingaporeMH619268MH619329985SingaporeMH619269MH6193301783Sumatra, IndonesiaMH619270MH6193311784Sumatra, IndonesiaMH619271MH6193321785Sumatra, IndonesiaMH619272MH619333MH6193852230Sulawesi, IndonesiaMH619273MH619334MH619386MH6194253343Bohol, PhilippinesMH619274MH619335MH619387MH6194263616Bohol, PhilippinesMH619275MH619336MH6193885627VietnamMH619276MH619337MH619389MH6194275646VietnamMH619277MH619338MH6193905958 HPeninsular MalaysiaMH619278MH619339MH619391MH6194285959Peninsular MalaysiaMH619279MH619340MH6193925960Peninsular MalaysiaMH619280MH619341MH6193935961Peninsular MalaysiaMH619281MH619342MH6193945963Peninsular MalaysiaMH619282MH619343MH6193955965Peninsular MalaysiaMH619283MH619344

Table 1. GenBank accession numbers for COI, 16S, ITS2, and 28S DNA sequences. All sequences are new, except the sequences of the outgroups which were obtained from our previous studies (Dayrat et al. 2011, 2016, 2017, 2018, 2019; Dayrat and Goulding 2017; Goulding et al. 2018a, b, c).SpeciesIndividual (DNA #)LocalityGenBank COIGenBank 16SGenBank ITS2GenBank 28SAlionchis jailoloensis5137Halmahera, IndonesiaMG953528MG953538MG953548MK122918Marmaronchis marmoratus5409Madang, Papua New GuineaMK122838MK122859MK122893MK122915Marmaronchis vaigiensis1183SingaporeMK122812MK122854MK122877MK122910Melayonchis aileenae970Peninsular MalaysiaKX240033KX240057MK122902MK125514Melayonchis eloisae1011SingaporeKX240026KX240050MK122904MK125515Onchidella celtic a5013FranceMG958715MG958717MK122906MK122921Onchidella nigricans1524New South Wales, AustraliaMG970878MG970944MK122908MK122923Onchidina australis1523New South Wales, AustraliaKX179548KX179561MG958719MG958887Onchidium stuxbergi5605VietnamKX179520KX179537MG958721MG958886Onchidium typhae965Peninsular MalaysiaKX179509KX179525MG958720MG958885Paromoionchis daemelii1511New South Wales, AustraliaMH055048MH055129MH055241MH055289Paromoionchis tumidus1732Sumatra, IndonesiaMH054951MH055104MH055196MH055268Peronia sp.696Okinawa, JapanHQ660043HQ659911MG958871MG958883Peronia sp.706Hawaii, USAHQ660038HQ659906MG958722MG958884Peronina tenera960Peninsular MalaysiaMG958740MG958796MG958840MG958874Peronina zulfigari6005Peninsular MalaysiaMG958775MG958831MG958867MG958882Platevindex luteus1001SingaporeMG958714MG958716MG958718MG958888Wallaconchis ater5121Halmahera, IndonesiaMG970820MG970911MG971134MG971186Wallaconchis sinanui2740Ambon, IndonesiaMG970713MG970881MG971093MG971161L. boucheti1004SingaporeMH619242MH6193031005SingaporeMH619243MH619304MH619364MH6194131037Brunei DarussalamMH619244MH619305MH6193651038Brunei DarussalamMH619245MH619306MH619366MH6194141679Northern Territory, AustraliaMH619246MH6193071681Northern Territory, AustraliaMH619247MH619308MH619368MH6194161685Northern Territory, AustraliaMH619248MH6193091688 HNorthern Territory, AustraliaMH619249MH6193101729Sumatra, IndonesiaMH619250MH619311MH6193692559Queensland, AustraliaMH619251MH619312MH6193702578Queensland, AustraliaMH619252MH619313MH6193712593Queensland, AustraliaMH619253MH619314MH6193722604Queensland, AustraliaMH619254MH619315MH619373MH6194172609Queensland, AustraliaMH619255MH619316MH619374MH6194182612Queensland, AustraliaMH619256MH619317MH6193752692Queensland, AustraliaMH619257MH619318MH619376MH6194192693Queensland, AustraliaMH619258MH6193193615Bohol, PhilippinesMH619259MH619320MH619377MH619420914Peninsular MalaysiaMH619260MH619321MH619378MH619421915Peninsular MalaysiaMH619261MH619322MH6193795520Peninsular MalaysiaMH619262MH619323MH6193805609VietnamMH619263MH619324MH6193815610VietnamMH619264MH619325MH619382MH619422L. bourkei bourkei1656Northern Territory, AustraliaMH619290MH6193511616Northern Territory, AustraliaMH619291MH619352MH619402MH6194321617Northern Territory, AustraliaMH619292MH619353MH6194031618Northern Territory, AustraliaMH619293MH6193541621Northern Territory, AustraliaMH619294MH619355MH6194041652Northern Territory, AustraliaMH619295MH619356MH6194051657 HNorthern Territory, AustraliaMH619296MH619357MH6194061659Northern Territory, AustraliaMH619297MH619358MH6194071666Northern Territory, AustraliaMH619298MH619359MH6194081673Northern Territory, AustraliaMH619299MH619360MH619409MH6194341692Northern Territory, AustraliaMH619300MH619361MH6194101693Northern Territory, AustraliaMH619301MH619362MH619411MH6194351694Northern Territory, AustraliaMH619302MH619363MH619412MH619436L. bourkei lateriensis2743Ambon, IndonesiaMH619284MH619345MH619396MH6194292753Ambon, IndonesiaMH619285MH619346MH619397MH6194306061Ambon, IndonesiaMH619286MH619347MH6193986063Ambon, IndonesiaMH619287MH619348MH6193996064 HAmbon, IndonesiaMH619288MH619349MH619400MH6194316065Ambon, IndonesiaMH619289MH619350MH619401L. bourkei matangensis978SingaporeMH619265MH619326MH619383MH619423979SingaporeMH619266MH619327MH619384MH619424980SingaporeMH619267MH619328983SingaporeMH619268MH619329985SingaporeMH619269MH6193301783Sumatra, IndonesiaMH619270MH6193311784Sumatra, IndonesiaMH619271MH6193321785Sumatra, IndonesiaMH619272MH619333MH6193852230Sulawesi, IndonesiaMH619273MH619334MH619386MH6194253343Bohol, PhilippinesMH619274MH619335MH619387MH6194263616Bohol, PhilippinesMH619275MH619336MH6193885627VietnamMH619276MH619337MH619389MH6194275646VietnamMH619277MH619338MH6193905958 HPeninsular MalaysiaMH619278MH619339MH619391MH6194285959Peninsular MalaysiaMH619279MH619340MH6193925960Peninsular MalaysiaMH619280MH619341MH6193935961Peninsular MalaysiaMH619281MH619342MH6193945963Peninsular MalaysiaMH619282MH619343MH6193955965Peninsular MalaysiaMH619283MH619344

Table 2. Intra- and inter-unit pairwise genetic distances between the four mitochondrial units of Laspionchis based on our data set of 61 COI sequences (Table 1). Ranges of minimum to maximum distances are indicated (as percentages): e.g., individual sequences within L. boucheti are between 0 and 2.7% divergent, and individual sequences between L. boucheti and L. bourkei bourkei are minimally 8.6% and maximally 10.4% divergent. Overall, the distance gap between all four mitochondrial units is between 2.5% (the maximum intra-unit distance within L. boucheti and within L. bourkei matangensis) and 3.9% (the minimum distance between L. bourkei lateriensis and L. bourkei matangensis). Finally, the distance gap between the two species L. boucheti and L. bourkei is between 2.5% and 7.5%.SpeciesL. bouchetiL. bourkeibourkeilateriensismatangensisL. boucheti0.0-2.5L. bourkei bourkei8.6-10.40.0-1.2L. bourkei lateriensis7.5-8.56.1-7.80.0-0.0L. bourkei matangensis7.5-9.55.3-6.13.9-5.50.0-2.5

Table 2. Intra- and inter-unit pairwise genetic distances between the four mitochondrial units of Laspionchis based on our data set of 61 COI sequences (Table 1). Ranges of minimum to maximum distances are indicated (as percentages): e.g., individual sequences within L. boucheti are between 0 and 2.7% divergent, and individual sequences between L. boucheti and L. bourkei bourkei are minimally 8.6% and maximally 10.4% divergent. Overall, the distance gap between all four mitochondrial units is between 2.5% (the maximum intra-unit distance within L. boucheti and within L. bourkei matangensis) and 3.9% (the minimum distance between L. bourkei lateriensis and L. bourkei matangensis). Finally, the distance gap between the two species L. boucheti and L. bourkei is between 2.5% and 7.5%.SpeciesL. bouchetiL. bourkeibourkeilateriensismatangensisL. boucheti0.0-2.5L. bourkei bourkei8.6-10.40.0-1.2L. bourkei lateriensis7.5-8.56.1-7.80.0-0.0L. bourkei matangensis7.5-9.55.3-6.13.9-5.50.0-2.5

Table 3. Summary of traits that can help distinguish the two species of Laspionchis. All traits are subject to individual variation. Traits are described in detail in the corresponding species descriptions. Traits are also indicated for the three subspecies of L. bourkei.SpeciesRetractor muscle (penis)Retractor muscle (penis) attachment siteDistal muscle fibersAccessory penial gland spine size (mm)Penial hooks (μm)DistributionL. bouchetiStrong and longPosterior end of visceral cavity (by the rectum)yes0.7 to 160 to 160Peninsular Malaysia, Indonesia (Sumatra), Singapore, Brunei, Vietnam, Philippines (Bohol), Australia (Northern Territory, Queensland)L. bourkei bourkeiVery shortAnterior third of the visceral cavityno0.75 to 120 to 35Australia (Northern Territory)L. bourkei lateriensisAbsent or vestigial-no0.35 to 0.7520 to 45Indonesia (Ambon)L. bourkei matangensisAbsent or vestigial-no0.43 to 0.5715 to 40Peninsular Malaysia, Indonesia (Sulawesi, Sumatra), Singapore, Philippines (Bohol), Vietnam

Table 3. Summary of traits that can help distinguish the two species of Laspionchis. All traits are subject to individual variation. Traits are described in detail in the corresponding species descriptions. Traits are also indicated for the three subspecies of L. bourkei.SpeciesRetractor muscle (penis)Retractor muscle (penis) attachment siteDistal muscle fibersAccessory penial gland spine size (mm)Penial hooks (μm)DistributionL. bouchetiStrong and longPosterior end of visceral cavity (by the rectum)yes0.7 to 160 to 160Peninsular Malaysia, Indonesia (Sumatra), Singapore, Brunei, Vietnam, Philippines (Bohol), Australia (Northern Territory, Queensland)L. bourkei bourkeiVery shortAnterior third of the visceral cavityno0.75 to 120 to 35Australia (Northern Territory)L. bourkei lateriensisAbsent or vestigial-no0.35 to 0.7520 to 45Indonesia (Ambon)L. bourkei matangensisAbsent or vestigial-no0.43 to 0.5715 to 40Peninsular Malaysia, Indonesia (Sulawesi, Sumatra), Singapore, Philippines (Bohol), Vietnam

Table 4. Radular formulae for the two species of Laspionchis, following the format number of rows × number of lateral teeth per left half row - 1 (rachidian tooth)- number of lateral teeth per right half row. Each DNA extraction number corresponds to one individual. The voucher catalog numbers can be shared by several individuals when collected at exactly the same locality (each individual is preserved in its own separate vial with its corresponding DNA number).SpeciesRadular formulaSpecimen length (mm)VoucherDNA extraction numberL. boucheti43 × 50-1-5030NTM P.576141688 H57 × 90-1-9031BDMNH103755 × 70-1-7031ITBZC IM 000175609L. bourkei bourkei55 × 75-1-7523NTM P.576151657 H50 × 75-1-7532NTM P.57618166645 × 65-1-6525NTM P.57619169243 × 60-1-6019NTM P.576181673L. bourkei lateriensis45 × 65-1-6517UMIZ 001156064 H50 × 50-1-5018UMIZ 00116606345 × 60-1-6015UMIZ 00116606540 × 55-1-5515UMIZ 001166061L. bourkei matangensis42 × 60-1-6015USMMC 000555958 H55 × 70-1-7025PNM 041254334345 × 60-1-6012USMMC 00056596041 × 55-1-5515USMMC 00056595937 × 45-1-458USMMC 000565965

Table 4. Radular formulae for the two species of Laspionchis, following the format number of rows × number of lateral teeth per left half row - 1 (rachidian tooth)- number of lateral teeth per right half row. Each DNA extraction number corresponds to one individual. The voucher catalog numbers can be shared by several individuals when collected at exactly the same locality (each individual is preserved in its own separate vial with its corresponding DNA number).SpeciesRadular formulaSpecimen length (mm)VoucherDNA extraction numberL. boucheti43 × 50-1-5030NTM P.576141688 H57 × 90-1-9031BDMNH103755 × 70-1-7031ITBZC IM 000175609L. bourkei bourkei55 × 75-1-7523NTM P.576151657 H50 × 75-1-7532NTM P.57618166645 × 65-1-6525NTM P.57619169243 × 60-1-6019NTM P.576181673L. bourkei lateriensis45 × 65-1-6517UMIZ 001156064 H50 × 50-1-5018UMIZ 00116606345 × 60-1-6015UMIZ 00116606540 × 55-1-5515UMIZ 001166061L. bourkei matangensis42 × 60-1-6015USMMC 000555958 H55 × 70-1-7025PNM 041254334345 × 60-1-6012USMMC 00056596041 × 55-1-5515USMMC 00056595937 × 45-1-458USMMC 000565965