Org Divers Evol (2018) 18:187–210 https://doi.org/10.1007/s13127-017-0352-4

ORIGINAL ARTICLE

Rolling into the deep of the land (Tricladida, , )

Fernando Carbayo1 & Marcos Santos Silva1 & Marta Riutort2 & Marta Álvarez-Presas2

Received: 27 June 2017 /Accepted: 1 November 2017 /Published online: 1 December 2017 # Gesellschaft für Biologische Systematik 2017

Abstract The land planarian genus Choeradoplana present a comprehensive phylogenetic tree including most (Plathelminthes, Tricladida) is currently integrated by 13 spe- Choeradoplana . The tree, well supported, allows mak- cies. In previous works, morphological variation in its type ing some preliminary inferences on the evolution of the group species, Choeradoplana iheringi, was reported, but no attempt and its historical biogeography. to test whether it is just a single species has been made yet. In order to disentangle the taxonomy of this species and further Keywords ABGD . bPTP . BP&P . Cryptic species . members of the genus, we sampled new specimens and com- . GMYC . Histology . bined morphological and molecular data and also have evalu- ated the performance of diverse methods of molecular species delimitation. Our data point to the presence of two cryptic spe- Introduction cies named C. iheringi, plus two new species, all hidden under the same general appearance. An in-depth morphological study In recent years, the use of molecular data has increased con- of the specimens allowed detection of diagnostic morphologi- siderably the discovery of cryptic species (considering cryptic cal traits in each species, for which we also propose a molecular species those morphologically indistinguishable but diagnosis. This integrative taxonomic study demonstrates once belonging to different evolutionary lineages; Waeschenbach again the usefulness of molecular tools to weigh minor mor- et al. 2012) and promoted their study (Bickford et al. 2007). phological characteristics and thus reveal the existence of spe- This increase is related to the need of a better knowledge of cies that would otherwise remain cryptic. However, under cer- species diversity in order to design good conservation plans tain parameters, the molecular methods may over-split species for endangered regions or for the control of invasive species, with a high genetic structure, maybe pointing to incipient spe- among other causes. For instance, if we are only aware of the ciation. This makes critical the use of these methods combined species at the morphotype level, we may be underestimating with a comprehensive morphological approach. We also the real biodiversity both at the taxonomic and genetic levels, which is important for the study of potential distribution Electronic supplementary material The online version of this article changes underwent by species as a result of climate change (https://doi.org/10.1007/s13127-017-0352-4) contains supplementary (Bálint et al. 2011). Generally, the use of the DNA barcoding material, which is available to authorized users. initiative (Hebert et al. 2003), based on a single short fragment of DNA, is sufficient to identify the species or even delimit * Marta Álvarez-Presas new ones, but in many cases, the use of other tools (more [email protected] morphological characters, more genes, or new molecular tools to delimit species) is necessary to the determination of evolu- 1 Laboratório de Ecologia e Evolução, Escola de Artes, Ciências e Humanidades, Universidade de São Paulo (USP), Av. Arlindo Bettio, tionary divergent taxonomic units (Prévot et al. 2013; 1000, CEP, São Paulo, SP 03828-000, Hamilton et al. 2014). 2 Departament de Genètica, Microbiologia i Estadística, Facultat de For terrestrial (Platyhelminthes, Tricladida, Biologia and Institut de Recerca de la Biodiversitat (IRBio), Continenticola, Geoplanidae), repeated cases of cryptic spe- Universitat de Barcelona, Av Diagonal, 643 08028 Barcelona, cies have been detected (Lemos et al. 2014; Álvarez-Presas 188 Carbayo F. et al. et al. 2015; Carbayo et al. 2016; Mateos et al. 2017). Thanks Previous studies that include this species already showed to the combination of different methodologies of molecular signs of the presence of cryptic species confounded under the species delimitation (GMYC and BP&P for instance) it has name C. iheringi. Riester (1938) and Leal-Zanchet and de been possible to focus efforts on the morphological character- Souza (2003) documented morphological variation in ization of molecularly differentiated lineages that until then C. iheringi mainly regarding the relative size of parts of the had been considered the same species, in an integrative tax- copulatory apparatus. This variation was interpreted as a result onomy approach. Thus, it has shown profitable for the taxon- of artifactual contraction, physiological condition, or popula- omy of this group the application of a protocol beginning with tional variation (Riester 1938; Leal-Zanchet and de Souza an external morphology assignation to morphospecies, subse- 2003). quent molecular testing of the morphospecies, and, when both More recently, Carbayo et al. (2013) provided a molecular approaches are not congruent, deeper morphological exami- phylogeny of the subfamily Geoplaninae, into which two nation (Álvarez-Presas et al. 2015; Carbayo et al. 2016; specimens of C.cf.iheringi were represented. One specimen Mateos et al. 2017). was collected in São Francisco de Paula, and another in the Geoplanids of the genus Choeradoplana Graff, 1896 are Serra da Bocaina National Park (SP), close to Salesópolis. generally easy to recognize by means of their characteristical- They noted the paraphyletic status of the species and sug- ly rolled-up and laterally expanded cephalic end. A peculiar gested that morphological differences, already highlighted characteristic of the representatives of this genus is the pres- by Leal-Zanchet and de Souza (2003), might be indicating ence of a very strong longitudinal muscular subepidermic lay- non-conspecificity. er that allows them, in an alarm state, to escape by curling Here, we present a comprehensive molecular phylogenetic upward the back end and roll forward until they touch the framework of 11 species of the genus, including numerous substrate again, resulting in a sort of somersault (Froehlich representatives of Choeradoplana cf. iheringi, to test the phy- 1955a). letic status of this species. The representativeness of the genus The genus is currently integrated by 13 species, all of them also encompasses two new cryptic species which can be con- with neotropical distribution (found in Brazil and ). fused with C. iheringi as regards of the chromatic pattern of Most species of Choeradoplana have been described and their dorsum. Finally, robustness of our phylogenetic trees delimited by means of morphological traits only. Molecular also allowed us to make considerations on the biogeography approaches to the taxonomy of the genus were briefly ad- of the genus. dressed by Carbayo et al. (2013). They suggested a polyphy- letic status for C. iheringi Graff, 1899 (Carbayo et al. 2013). More recently, C. minima Lemos & Leal-Zanchet, 2014 and Material and methods C. benyai Lemos & Leal-Zanchet, 2014 (Lemos et al. 2014) were described based on morphological and molecular data Morphological analyses (Lemos et al. 2014). No further taxonomic molecular works have been issued. We examined the type material of C. iheringi, and most spec- Morphological aspects of species of the genus are unevenly imens studied by Riester (1938), Marcus (1951), Froehlich studied, ranging from C. ehrenreichi Graff, 1899—the internal (1955b), and Leal-Zanchet and de Souza (2003) and further organs of which are unknown—to Choeradoplana iheringi, individuals collected recently by us. Graff’s material consists the type species of the genus, which was studied by several of sagittal and transverse sections of six cephalic portions, authorities. This species was originally described from several sagittal sections of a pharynx, sagittal sections of a copulatory specimens collected in Taquara (State of Rio Grande do Sul apparatus, and sagittal-to-horizontal sections of a twisted por- (RS)) (Fig. 1)(Graff1899). Riester (1938) studied additional tion of the body that includes pharynx and copulatory appa- material collected in Gorduras (State of Minas Gerais (MG)), ratus. We reconstructed the copulatory apparatus of the spec- Teresópolis (State of Rio de Janeiro (RJ)), and Riberião Pires imen described and illustrated by Graff (1899, Fig. 45, p. 194). (State of São Paulo (SP)). From the latter locality, Marcus Riester’s material consists of several specimens having (1951) and Froehlich (1955b) examined further specimens. some parts of their bodies preserved in balsam and other parts Froehlich (1956) also reported more specimens from sectioned on histological slides. We took microphotographs of Teresópolis bearing a chromatic pattern of the dorsum similar the four copulatory apparatuses available and analyzed them. to that described in Riester (1938). Leal-Zanchet and de Souza The copulatory apparatus of the specimens N653 and N83 do (2003) redescribed the species by examining individuals from not match the general anatomy of the nominal C. iheringi and São Francisco de Paula (RS) and Salesópolis (SP). Regarding were not further considered here. their ecology, the species prefers altered habitats so that they Marcus’ material consists of sagittal sections of the phar- can be a good indicator of biological disturbance in the habitat ynx (on one slide) and of the copulatory apparatus (two slides) in which they are present (Carbayo et al. 2002). belonging to the collection of late Prof. E. M. Froehlich and Rolling into the deep of the land planarian genus Choeradoplana (Tricladida, Continenticola, Geoplanidae)... 189

Fig. 1 Distribution map of the four species of Choeradoplana described in this paper. (The high version of this figure can be found in the Electronic Supplementary Material)

provisionally under the care of FC. We analyzed this material https://www.ral-farben.de/uebersicht-ral-classic-farben.html? under a light microscope. &L=1). Type specimens are deposited in the Museu de Froehlich’s material consists on sections of anterior end of Zoologia da Universidade de São Paulo (MZUSP). the body; as we were unable to identify the material, it was not further considered in this work. We also studied histological Molecular analyses sections of the copulatory apparatus of the specimens MZU PL00026, MZUSP PL 147, and MZUSP PL 148 of Samples preserved in 100% ethanol were used for DNA ex- C. iheringi analyzed by Leal-Zanchet and de Souza (2003). traction with the Wizard® Genomic Purification Kit Finally, aimed at a comprehensive molecular phylogenetic (Promega, Madison, WI, USA) following the same protocol tree and morphological studies, we also collected putative rep- as in Álvarez-Presas et al. (2011). A fragment of approximate- resentatives of C. iheringi, and other species in several locali- ly 1 kb of the gene encoding the mitochondrial cytochrome ties in South East and South Brazil (Table 1). We searched for oxidase I (Cox1) was amplified by polymerase chain reaction these on the soil litter and forest trails during the day (PCR) following the same PCR conditions and primers as in and at night. We took pictures of them, euthanized in boiling Álvarez-Presas et al. (2011). Moreover, four nuclear genes water, cut off a small tissue sample, and preserved it in 100% (ribosomal 18S type II (~1700 bp), 28S (~1600 bp), and ethanol for DNA extraction. We fixed in 10% formalin the ITS-1 (~500 bp) and elongation factor 1-alpha (EF; main part of the body and, subsequently, transferred it to 80% ~700 bp) were amplified. For 18S and 28S, we used primers ethanol. Parts of the body were embedded in paraffin and PCR conditions as in Álvarez-Presas et al. (2008), for Histosec®, sectioned at 3–7 μm, transferred to glass slides, ITS-1asinÁlvarez-Presasetal.(2011), and for the EF gene and subsequently stained with the Mallory method as modified as in Carbayo et al. (2013). The same primers were used for by Cason (1950). We examined the slides and reconstructed the PCR amplification and sequencing. PCR reactions were per- copulatory apparatuses and pharynges with a compound micro- formed in a final volume of 25 μL with final concentrations of scope and a camera lucida attached. We took digital micropho- 0.5× Go Taq® Flexi buffer (Promega), 2 mM MgCl2,0.1mM tographs with the help of a digital camera attached to the mi- each dNTP, 1 mM of each primer, 0.75 units of Taq DNA croscope and edited them with GIMP (GNU Image Polymerase Go® (Promega), and DNA template. The ampli- Manipulation Program 2.8.10; The GIMP team www.gimp. fication products were purified directly with a vacuum pump org,1995–2014) so as to enhance the contrast and provide a (Multiscreen®HTS Vacuum Manifold, Millipore whitish background. The width of the creeping sole was Corporation, Billerica, MA, 01821, USA) or with measured on transverse sections of the pre-pharyngeal region. exonuclease/shrimp alkaline phosphatase (EXOSAP). DNA Representatives of species studied and not described in this sequences were determined from both chains by Sanger se- paper were identified by the external aspect of their bodies and quencing in Macrogen (Amsterdam, ) or in the Unitat the morphology of the copulatory apparatuses (Table S1). de Genòmica dels Serveis Científico-Tècnics of the Color descriptions of the body follow the online palette Universitat de Barcelona (Barcelona, Spain), using Big-Dye RAL colors (© RAL Gemeinnützige GmbH, available at (3.1, Applied Biosystems, Foster City, CA, USA) and an 190 Table 1 List of Choeradoplana samples used in this study with sampling locality, molecular code, voucher code, and GenBank accession numbers

GenBank accession number Species Collecting locality Field number Museum code Cox1 18S 28S EF ITS Choeradoplana abaiba sp. nov. P.E.S. do Tabuleiro/SC F3221 MZUSPb PL 595 HQ026428 MF802572 MF802616 MF802667 HQ026475 P.E.S. do Tabuleiro/SC F3240 MZUSP PL 503 MF802627 MF802573 MF802617 – MF802676 P.E.S. do Tabuleiro/SC F3270 MZUSP PL 505 MF802628 MF802574 MF802618 MF802668 MF802677 P.E.S. do Tabuleiro/SC F3312 MZUSP PL 509 MF802629 MF802575 ––MF802678 P.E.S. do Tabuleiro/SC F3315 MZUSP PL 511 HQ542891 MF802576 ––MF802679 P.E.S. do Tabuleiro/SC F3864 MZUSP PL 1166 MF802630 MF802577 ––MF802680 P.E.S. do Tabuleiro/SC F3865 MZUSP PL 1167 MF802631 MF802578 ––MF802681 P.E.S. do Tabuleiro/SC F3866 MZUSP PL 1168 MF802632 MF802579 ––MF802682 P.E.S. do Tabuleiro/SC F3894 MZUSP PL 1169 MF802633 MF802580 ––MF802683 P.E.S. do Tabuleiro/SC F3905 MZUSP PL 1170 MF802634 MF802581 ––MF802684 Choeradoplana agua sp. nov. P.E. do Desengano/RJ F2204 MZUSP PL 445 MF802635 MF802582 ––MF802685 P.E. do Desengano/RJ F2205 MZUSP PL 446 MF802636 MF802583 ––MF802686 P.E. do Desengano/RJ F2214 MZUSP PL 448 MF802637 MF802584 ––MF802687 P.E. do Desengano/RJ F3952 MZUSP PL 619 KF971680 MF802585 MF802619 MF802669 MF802688 Choeradoplana albonigra R. B. Augusto Ruschi/ES F2313 MZUSP PL 1109 KF971684 –––MF802689 (Riester, 1938) R. B. Augusto Ruschi/ES F2391 MZUSP PL 1113 KF971683 MF802586 MF802620 – MF802690 P.E. do Desengano/RJ F4081 MZUSP PL 1083 KC608327 KC608561 KC608444 KC614545 MF802691 Choeradoplana banga Carbayo P.E.S. da Cantareira/SP F2023 MZUSP PL 1000 KC608267 KC608499 KC608382 –– & Froehlich, 2012 P.E.S. da Cantareira/SP F3006 MZUSP PL 1001 MF802638 MF802587 MF802621 MF802670 MF802692 P.E.S. da Cantareira/SP F3011 MZUSP PL 1002 KC608301 KC608534 KC608417 KC614523 MF802693 P.E.S. da Cantareira/SP F3706 MZUSP PL 568 MF802639 –––– Campos de Jordão/SP F4581 MZUSP PL 1175 – MF802588 ––MF802694 Choeradoplana benyai Lemos FLONA-SFPa/RS F3494 MZUSP PL 1163 MF802640 MF802589 MF802622 MF802671 MF802695 & Leal-Zanchet, 2014 FLONA-SFP/RS Chosp2–151 c MZU PL.00151 KJ690049 –––KJ690064 FLONA-SFP/RS Chosp2–152 c MZU PL.00152 KJ690050 –––KJ690065 P.E.S. do Tabuleiro/SC F3813 MZUSP PL 1165 MF802641 MF802590 MF802623 MF802672 MF802696 Choeradoplana bocaina Carbayo P.N.S. da Bocaina/SP F2104 MZUSP PL 999 KC608273 KC608505 KC608388 KC614500 MF802697 & Froehlich, 2012 P.N.S. da Bocaina/SP F2803 MZUSP PL 998 KC608283 KC608515 KC608398 KC614509 MF802698 P.N.S. da Bocaina/SP F2822 MZUSP PL 997 KC608288 KC608520 KC608403 – MF802699 Choeradoplana gladismariae P.E. Intervales/SP F3092 MZUSP PL 1063 KC608306 KC608540 KC608423 KC614528 MF802700 Carbayo & Froehlich, 2012 P.E. Intervales/SP F3802 MZUSP PL 1004 KC608326 KC608560 KC608443 –– aby .e al. et F. Carbayo Choeradoplana iheringi Parobé/RS F0365 MZUSP PL 651 MF802642 MF802591 MF802624 MF802673 MF802701 Graff 1899 FLONAa-SFP/RS F3355 MZUSP PL 512 MF802643 MF802592 ––– FLONA-SFP/RS F3362 MZUSP PL 514 MF802644 MF802593 ––MF802702 FLONA-SFP/RS F3369 MZUSP PL 516 MF802645 MF802594 ––MF802703 oln notede fteln lnra genus planarian land the of deep the into Rolling Table 1 (continued) FLONA-SFP/RS F3398 MZUSP PL 519 MF802646 –––MF802704 FLONA-SFP/RS F3400 MZUSP PL 521 HQ026429 MF802595 ––HQ026476 FLONA-SFP/RS F3401 MZUSP PL 522 MF802647 MF802596 ––MF802705 FLONA-SFP/RS F3407 MZUSP PL 523 MF802648 MF802597 ––MF802706 FLONA-SFP/RS F3409 MZUSP PL 524 MF802649 MF802598 ––MF802707 FLONA-SFP/RS F3412 MZUSP PL 525 MF802650 MF802599 ––MF802708 FLONA-SFP/RS F3430 MZUSP PL 528 MF802651 MF802600 ––MF802709 FLONA-SFP/RS F3445 MZUSP PL 529 MF802652 MF802601 ––– FLONA-SFP/RS F3446 MZUSP PL 530 MF802653 MF802602 ––MF802710 FLONA-SFP/RS F3447 MZUSP PL 531 MF802654 MF802603 ––MF802711 FLONA-SFP/RS F3448 MZUSP PL 532 MF802655 MF802604 ––MF802712 FLONA-SFP/RS F3449 MZUSP PL 533 KC608320 KC608554 KC608437 KC614540 MF802713 FLONA-SFP/RS F3450 MZUSP PL 1160 MF802656 MF802605 ––MF802714 FLONA-SFP/RS F3451 MZUSP PL 534 MF802657 MF802606 ––MF802715

FLONA-SFP/RS F3454 MZUSP PL 535 MF802658 MF802607 ––MF802716 Choeradoplana FLONA-SFP/RS F3461 MZUSP PL 1161 MF802659 MF802608 ––MF802717 FLONA-SFP/RS F3465 MZUSP PL 537 MF802660 MF802609 ––MF802718 FLONA-SFP/RS F3479 MZUSP PL 538 MF802661 MF802610 ––MF802719 FLONA-SFP/RS F3481 MZUSP PL 539 MF802662 MF802611 ––MF802720 FLONA-SFP/RS F3491 MZUSP PL 1162 MF802663 MF802612 ––MF802721 Tildd,Cnietcl,Golnde..191 Geoplanidae)... Continenticola, (Tricladida, FLONA-SFP/RS F3495 MZUSP PL 1164 MF802664 –––– FLONA-SFP/RS Ciherin155c MZU PL.00155 KJ690045 –––KJ690055 FLONA-SFP/RS Ciherin156 c MZU PL.00156 KJ690046 –––KJ690056 FLONA-SFP/RS Ciherin157 c MZU PL.00157 KJ690047 –––– FLONA-SFP/RS Ciheringi1 c MZU PL.00158 KJ690048 –––– Choeradoplana marthae E.B. Boraceia/SP F2137 MZUSP PL 1153 MF802665 MF802613 MF802625 MF802674 MF802722 Froehlich, 1955 Choeradoplana minima Lemos FLONA-SFP/RS Chosp1–143 c MZU PL.00143 KJ690052 –––KJ690067 & Leal-Zanchet, 2014 FLONA-SFP/RS Chosp1–145 c MZU PL.00145 KJ690051 –––KJ690066 Choeradoplana pucupucu P.N.S. da Bocaina/SP F2840 MZUSP PL 540 KC608293 KC608525 KC608408 KC614517 MF802723 sp. nov. P.N.S. da Bocaina/SP F2844 MZUSP PL 541 MF802666 MF802614 MF802626 MF802675 MF802724 São José do Barreiro/SP F2847 MZUSP PL 1158 – MF802615 ––MF802725 Outgroup Matuxia matuta (Froehlich, 1955) P.E. Desengano/RJ F2184 MZUSP PL 1021 KC608276 KC608508 KC608391 KC614503 – P.E. Desengano/RJ F2187 MZUSP PL 1022 KC608277 KC608509 KC608392 KC614504 MF817452 Matuxia tuxaua (Froehlich, 1955) P.E.S. da Cantareira/SP F3059 MZUSP PL 1058 KC608302 KC608536 KC608419 KC614525 MF802726 Matuxia sp. FLONA-SFP/RS Matuxia166 c MZU PL.00166 KJ690054 –––KJ690069 FLONA-SFP/RS Matuxia167 c MZU PL.00167 KJ690053 –––KJ690068

See also Table S1 for field data and identification details a Floresta Nacional de São Francisco de Paula b Vouchers are deposited in the Museu de Zoologia da Universidade de São Paulo (MZUSP) c Sequences from Lemos et al. (2014) 192 Carbayo F. et al. automated sequencer ABI Prism 3730. Contigs were assem- partitions in the ML analyses. Trees were visualized and bled in Geneious 8.1.7 software (www.geneious.com;Kearse edited using FigTree v1.4.3 (Rambaut 2007-2017). et al. 2012). For the molecular species delimitation analyses, we ap- Since Cox1 and EF are coding genes, sequences were plied three discovery methods and one validation method. aligned basing on the amino acid sequences. We obtained a First, we implemented BI under a lognormal relaxed clock multiple alignment with Clustal W (included in the BioEdit on an arbitrary timescale, using BEAST v1.8.2 (Drummond software 7.0.9.0 (Hall 1999)). Ribosomal RNA gene se- et al. 2012) with the Cox1Del dataset and the GTR + I + Γ quences were aligned using the online version of the software model for 150 million generations. Tracer v1.6 (Rambaut et al. Mafft v7 (Katoh and Standley 2013) applying the G-INS-i 2014) was used to check that the parameters had reached iterative refinement method and, subsequently, checked the values of effective sample size (ESS) over 100. The default alignments by eye with BioEdit. Gblocks v0.91b program 10% were used as burn-in in TreeAnnotator v1.7.5 (Talavera and Castresana 2007) was used to eliminate objec- (Drummond and Rambaut 2007). The tree obtained was visu- tively misaligned or ambiguously aligned positions. Based on alized with FigTree v1.4.3, converted to newick format, and these alignments, we estimated the DNA sequence evolution then submitted to the GMYC web server (Zhang et al. 2013) model that better fits the data by using jModelTest v2.1.4 to delimit the species, running the original R implementation (Darriba et al. 2012), applying the Akaike information criteri- of the GMYC model (Fujisawa and Barraclough 2013). on (AIC). Three different datasets have been used for several Using the same dataset (Cox1Del), the Automatic Barcode analyses: (1) Cox1Del dataset (64 sequences, 822 bp), includ- Gap Discovery (ABGD) method was applied (Puillandre et al. ed Cox1 sequences and was used for the GMYC, ABGD, and 2012) through the website http://wwwabi.snv.jussieu.fr/ bPTP analyses; (2) BPP datasets 18S, 28S, Cox1, ITS-1, and public/abgd/abgdweb.html. Instead of using one or more EF with 65 sequences (completing some sequences with miss- predefined distance thresholds to delimit species, the ABGD ing data (Ns); see Table 1), the information of each molecule method evaluates the intraspecific divergence by detecting separately was used to apply the BPP method; and (3) breaks in the distribution of pairwise genetic distances (the concatenated dataset (65 Choeradoplana sequences plus 5 Bbarcode gap^) and tries to directly determine the threshold outgroups from the genus Matuxia, 5069 bp), included the that is optimal for a given dataset (Fontaneto et al. 2015). If a information of the five genes (18S, 28S, EF, ITS-1, and satisfactory threshold is not detected, it is concluded that all Cox1) used to infer a general phylogeny; this alignment in- the sequenced specimens are conspecific. Through the use of a cludes only those animals for which we had at least three sliding window, a local slope function is used to identify the genes sequenced, or at least two for those coming from first statistically significant peak where the barcode gap rep- GenBank. In those cases, the rest of sequences were filled with resents a sudden increase in slope. The dataset is re-distributed Ns. recursively into thinner clusters until no more gaps can be We calculated pairwise genetic distances between some detected. In this study, since the program is optimized for genetic lineages under the Kimura-2-Parameters correction use with the COI gene, the default values of Pmin = 0.001 using the software MEGA v7.0.21 (Kumar et al. 2016). and Pmax = 0.10, steps = 10, and Nb bins = 20 were used. We Phylogenies of the concatenated datasets were inferred tested two different values of relative gap width (X), 1.5 (the through the Bayesian inference (BI) method using the soft- default barcoding gap value) and 0.75 (half of the default ware MrBayes v3.2.2 (Ronquist et al. 2012), implementing value), in order to increase the sensitivity of the analysis the appropriate evolutionary model according to jmodeltest (Puillandre et al. 2012), and the distance matrix was corrected results. We allowed partitions to evolve under different under the parameters of the K80 Kimura model with a models (GTR + I + Γ forCox1,HKY+I+Γ for 18S, GTR MinSlope = 1.5. + Γ for 28S and EF genes, and HKY + Γ for ITS-1) with The third discovery species delimitation method that we unlinked rates. Two runs were applied producing 5 million applied was the Bayesian implementation of the Poisson generations for each, and of these, 5000 trees were stored. A Tree Processes (PTP) bPTP (Zhang et al. 2013). In PTP, 25% default burn-in were used, likelihood values (log likeli- Yule-coalescence transition points are modeled based on the hood) of cold chains were checked to have reached station- change in substitution rates in the phylogenetic tree used as arity, and the convergence of the two runs was verified by the input. Therefore, it only requires a phylogenetic tree where the average standard deviation of split frequencies being less than branch lengths represent number of substitutions as input. The 0.01. A consensus tree from the remaining trees was obtained. delimitation of species was performed using the web server We also used the maximum likelihood (ML) method to infer bPTP (available at http://species.h-its.org/ptp/) using as input phylogenies with the software RAxML v8 (Stamatakis 2014) tree the output of RaxML obtained with the Cox1Del dataset. with bootstrap support values (Felsenstein 1985) calculated Default options and 300,000 MCMC generations were used. from 10,000 replicates. Due to the constraints of the program, To verify the reliability of the results, the convergence of each the GTRGAMMA evolutionary model was applied to all test was verified by visual checking of the likelihood plot. Rolling into the deep of the land planarian genus Choeradoplana (Tricladida, Continenticola, Geoplanidae)... 193

For the validation step, we applied a multilocus Bayesian for each of them. However, C. abaiba sp. n., like C. iheringi, species delimitation method (Yang and Rannala 2010; Yang is divided into two entities (C and D). and Rannala 2014) implemented in software BP&P v3.3 Results from initial partitions in ABGD (Fig. 2 in orange) (Yang 2015). Through the use of reversible-jump MCMC show that the number of groups ranges from 15 (when prior (rjMCMC), we test different species delimitation hypotheses maximal distance (P) is 0.001) to 10 (P = 0.0359). Recursive and estimate the posterior probability (PP) of each model. We partitions, in contrast, range from 29 groups (P =0.0010)to11 used the previous GMYC species assignation as starting hy- (P = 0.0359). The relative gap width (Supp. Fig. 2)onlyhad pothesis input for the BP&P analysis (with the exception of an influence on the number of groups assigned in minimum the individuals MZUSP PL 1158 and MZUSP PL 1175 (see and maximum partitions, giving exactly the same results as for Table 1) that were assigned by morphological identification the intermediate initial and recursive partitions. The results of rather than Cox1 sequences due to the lack of these). Also, a ABGD and bPTP (Fig. 2 in purple) are exactly the same, but guide-tree generated by 100 million generations in *BEAST differ from GMYC in considering A and B (C. iheringi)asa v1.8.4. (Drummond et al. 2012) with the five datasets (BP&P single species, and in assigning individual MZUSP PL 1083 18S, 28S, EF, ITS-1, and Cox1), applying the evolutionary (C. albonigra (Riester, 1938)) as a different species. model for each gene resulting from the previous jmodeltest Results of the validation step, the BP&P analysis (Fig. 2 in analysis, was used as input. We parameterized both ancestral blue and Supp. Fig. 3), show a topology obtained by the population size (theta, θ) and the time of origin for each spe- Bayesian method with *BEAST equivalent to that obtained cies (tau, Ƭ) with two different models: M1 for very large with MrBayes using the concatenated dataset, with a single populations and deep divergences with the gamma prior G difference, C. gladismariae Carbayo and Froehlich, 2012 is (2, 10) and M2 for small ancestral and shallow divergences sister to C. albonigra in *BEAST (Supp. Fig. 3). The results G (2, 2000). We used algorithm 0 and ran the rjMCMC anal- obtained when testing the entities delimited by GMYC sup- ysis in 100,000 generations (with a sampling interval of 2) port the presence of 11 species. The results for the two models excluding 10% as burn-in, and to test the robustness of the tested with different parameters (M1 and M2) are equivalent, results, we repeated these runs using different starting seeds. with no major variations in any of the PP values of the nodes For the final assignment of species, we followed an inte- (Supp. Fig. 3; numbers shown over and below the nodes). grative criterion, taking into account the molecular and mor- Some nodes of the guide-tree have been collapsed into fewer phological results. Whenever there was a conflict between species with respect to GMYC species assignation. This is the molecular and morphological perspectives, we gave prefer- case of the species C. benyai for which the results of GMYC ence to the latter and discussed these decisions. split individuals into two different species that are grouped in BP&P (with a PP for the node of 0.21–0.29). The two different assignations for C. bocaina (L and M) are also collapsed into one single species (PP = 0.66–0.74). With regard to the study Results species in this work, C. iheringi turns out to be a single species (clades A and B, PP = 0.71–0.75), as well as C. abaiba sp. n. Molecular results (clades C and D, PP = 0.04–0.02). In summary, the molecular delimitation of species shows a Molecular delimitation of species few differences among methodologies, but in all cases, the described species are clearly delimited. Figure 2 also shows a schematic summary of the results ob- tained in molecular species delimitation analyses. The GMYC Phylogenetic analysis analysis (Fig. 2 in red and Supp. Fig. 1) indicated a total of 12 clusters and 15 entities with a confidence interval of 6–15 with The final length of the concatenated dataset is 5069 bp (being the single method. The log likelihood at the optimal threshold the individual gene dataset lengths 1631 bp (18S), 1552 bp (431.7163) was significantly better than the null model of a (28S), 822 bp (Cox1), 449 bp (ITS-1), and 615 bp (EF)). single coalescent (426.4597) in a likelihood ratio test Figure 2 shows that all the species included in the analysis (p < 0.01). Five of these groups coincide with the species al- are monophyletic. The species relationships are recovered ready described prior to this study, while the species with high support, presenting all nodes high values in both C. iheringi, C. benyai Lemos and Leal-Zanchet, 2014, and methods (BI and ML). In this phylogeny, two main clades C. bocaina Carbayo and Froehlich, 2012 appear as two sepa- appear, the clade A-J and the clade K-O (Fig. 2). In the first rate entities (clades A and B, E and F, and L and M, respec- one, C. benyai is the sister group of the main species group of tively). The new species described in this study (C. pucupucu this study (C. iheringi and C. abaiba sp. n.), and at the same sp. n. and C. agua sp. n.) appear each as an entity (H and N, time, this group is sister clade to the species C. marthae respectively), coinciding with the proposal of a new species Froehlich, 1955, C. pucupucu sp. n., and C. banga Carbayo 194 Carbayo F. et al.

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Fig. 2 Summarized results from all species delimitation analyses and phylogeny represent the hypothesized species groupings based on morphological assignation of samples, represented on a maximum multiple analyses. The scale bar indicates the substitutions per site. likelihood tree resulting from analysis of the concatenated dataset. Inserted images C. abaiba sp. n. (a), C. iheringi (b), C. pucupucu sp. n. Labels correspond to the field codes of the specimens. Values at nodes (c), and C. agua sp. n (d). (The high version of this figure can be found in correspond to posterior probability (BI) and bootstrap (ML). Red letters the Electronic Supplementary Material) correspond to GMYC entity assignation. Colored bars to the right of the and Froehlich, 2012. In the basal position of this big clade, the Taxonomic section species C. minima Lemos and Leal-Zanchet, 2014 is situated, with a long branch that could be due to the high content of Choeradoplana iheringi Graff, 1899 missing data in the dataset for this species (since only the sequences in GenBank were available). Thus, although the Choeradoplana iheringi:Riester(1938), in part; Leal-Zanchet position of this species is supported with maximum values, and de Souza (2003), in part; Carbayo et al. (2013). this location could be artifactual. However, without the se- Material studied. Syntype NHM B 2775: Taquara (State quencing of the missing genes, we cannot confirm this hy- of Rio Grande do Sul, Brazil), Coll. H. v. Ihering, sagittal sec- pothesis. The second clade is composed by C. gladismariae tions of pharynx and copulatory apparatus on 20 slides. Pharynx and its sister group was formed by C. albonigra, C. bocaina, (of specimens MZUSP PL 23, PL 134, and 148) and copulatory and C. agua sp. n. apparatus (specimens MZUSP PL 147, MZUSP PL 148,and MZU PL 26), from Floresta Nacional de São Francisco de Morphological results Paula, São Francisco de Paula (State of Rio Grande do Sul, Brazil), previously studied by Leal-Zanchet and de Souza The morphological analyses of the species (green bar in (2003). MZUSP PL 651 (field number F0365): Parobé (Rio Fig. 2) result in a delimitation congruent with the valida- Grande do Sul, Brazil), approximately −29.63, −50.87, Coll. F. tion molecular delimitation methodology (BP&P), but in Carbayo et al., 29 May 2001, sagittal sections of pharynx and some cases with limited results. For instance, it has not copulatory apparatus on 9 slides, remaining body in 80% etha- been possible to check the taxonomic status of C. benyai nol. The following specimens were collected in Floresta (separated in clades E and F by all Cox1-based methods, Nacional de São Francisco de Paula, São Francisco de Paula but considered a single species by BP&P) because of (State of Rio Grande do Sul, Brazil): MZUSP PL 519 (field defective histological slides of the only representative of number F3398): −29.427361, −50.398389, Coll. F. Carbayo clade F. See also the cryptic species section in the general et al., 21 January 2009, in 80% ethanol. MZUSP PL 533 (field discussion. number F3449): −29.421111, −50.396500, Coll. F. Carbayo Rolling into the deep of the land planarian genus Choeradoplana (Tricladida, Continenticola, Geoplanidae)... 195 et al., 25 January 2009, sagittal sections of pharynx and copula- Geographic distribution and habitat Municipalities of tory apparatus on 37 slides, remaining body in 80% ethanol. Taquara, Parobé, São Francisco de Paula (State of Rio MZUSP PL 521 (field number F3400): −29.427361, Grande do Sul, Brazil). The species was found in Atlantic −50.398389, Coll. F. Carbayo et al., 21 January 2009, sagittal forests, in exotic forest plantations, and human-modified sections of pharynx and copulatory apparatus on 12 slides, re- environs. maining body in 80% ethanol. MZUSP PL 516 (field number F3369): −29.428944, −50.392000, Coll. F. Carbayo et al., 20 January 2009, sagittal sections of pharynx on 20 slides and Description sagittal sections of copulatory apparatus on 14 slides, remaining body in 80% ethanol. MZUSP PL 512 (field number F3355): Living adult animals up to 55 mm in length and 4 mm in width −29.428944, −50.392000, Coll. F. Carbayo et al., 20 January when creeping (Fig. 3a, b). Body slender, subcylindrical in 2009, sagittal sections of copulatory apparatus on 16 slides, cross section; at rest, it can become flattened. Cephalic region remaining body in 80% ethanol. MZUSP PL 1162 (field num- rolled up so that ventral surface, provided with glandular cush- ber F3491): −29.421111, −50.396500, Coll. F. Carbayo et al., 27 ions typical of the genus, is facing up; posterior end pointed. January 2009, transverse sections of pre-pharynx on 5 slides, General aspect ranging from light to dark brown. Ground sagittal sections of pharynx on 17 slides and sagittal sections color of dorsum cream to orange brown, covered with numer- of copulatory apparatus on 13 slides, anterior and posterior por- ous olive brown speckles. Speckles frequently merge with tions in 80% ethanol. MZUSP PL 514 (field number F3362): each other except for small rounded areas that show the −29.428944, −50.392000, Coll. F. Carbayo et al., 20 January ground color. A tenuous midline with ground color is gener- 2009, transverse sections of pre-pharynx on 11 slides, sagittal ally present. Anterior end pale. Ventral surface oyster white, sections of pharynx on 33 slides and sagittal sections of copula- with the exception of the orange-brown or beige-brown glan- tory apparatus on 17 slides, anterior and posterior portions in dular cushions. Creeping sole as wide as 81–90% of body 80% ethanol. MZUSP PL 524 (field number F3409): width. Mouth positioned at a distance from anterior end equal −29.436278, −50.373694, Coll. F. Carbayo et al., 22 January to 48–67% of body length; gonopore, at 70–82%. Eyes 2009, transverse sections of cephalic region on 23 slides, hori- formed by one pigmented cup with 40 μmindiameter.No zontal sections of ovaries region on 14 slides, transverse sections clear halos around them. Eyes absent in the very anterior end of pre-pharynx on 11 slides, sagittal sections of pharynx on 14 of the body. Posteriorly, they are irregularly arranged in a slides and sagittal sections of copulatory apparatus on 17 slides, marginal row of 3–4 eyes throughout the body length. posterior end in 80% ethanol. MZUSP PL 539 (field number Sensory pits, 20 μm deep, distributed ventro-laterally in a F3481): −29.420556, −50.393889, Coll. F. Carbayo et al., 27 uniserial row, initiating from 0.8 to 1.7 mm (or 2–4% of body January 2009, transverse sections of cephalic region on 18 length) behind the anterior end. At the pre-pharyngeal region, slides, horizontal sections of ovaries region on 15 slides, trans- most conspicuous glands are rhabditogenic, which pierce verse sections of pre-pharynx on 6 slides, sagittal sections of abundantly dorsal and marginal epidermis. This type of cells pharynx on 14 slides and sagittal sections of copulatory appara- is scarce on the ventral epidermis. Glandular margin absent. tus on 20 slides, posterior end in 80% ethanol. Ventral nerve plate with 70 μminthickness. The cutaneous musculature comprises three layers, namely, Type locality Taquara, State of Rio Grande do Sul, South a subepithelial circular layer followed by two diagonal layers Brazil. with decussate fibers, and then a strongly developed longitu- dinal layer. The pre-pharyngeal region is 50 μm thick dorsally, Diagnosis Species of Choeradoplana with dorsal color pat- and 100 μm ventrally, and its fibers are packed in dense bun- tern mottled with brownish speckles. CMI, 15–27%. Sperm dles (90–130 fibers each) dorsally, and loose bundles (10–30 ducts penetrate antero-ventral wall of penis bulb. Copulatory fibers each) ventrally (MZUSP PL 1162). As characteristic of apparatus relatively compact. Prostatic vesicle intrabulbar, the genus, part of the muscle fibers of the ventral longitudinal with proximal half canalicular and oriented dorsally. No per- layer are sunken into the parenchyma. CMI, 15–27%. In the manent penis papilla. Male:female atrial length ratio, 1.6–2.9. cephalic region, sunken cutaneous longitudinal muscle fibers gather medianly forming a retractor muscle, unroller of whole Molecular diagnosis This species includes all populations extremity, as described elsewhere (Carbayo and Leal-Zanchet that cluster with individuals MZUSP PL 512, MZUSP PL 2003). 514, MZUSP PL 516, MZUSP PL 519, MZUSP PL 521– Three usual parenchymal muscle layers are present 25, MZUSP PL 528–35, MZUSP PL 651, MZUSP PL 537– throughout the body: a well-developed layer of diagonal 39, MZUSP PL 1160–62, MZUSP PL 1164, and MZU PL deccusate fibers (25 μm thick), a transverse supraintestinal 155–58 (Table 1) from this study, with significant support in layer (50–80 μm), a transverse subintestinal one (50 μm) an adequate molecular delimitation model. (MZUSP PL 1162). An additional, subneural fourth layer of 196 Carbayo F. et al.

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Fig. 3 Choeradoplana iheringi Graff 1899. a Specimen MZUSP PL 651 view. d Diagrammatic representation of the copulatory apparatus of creeping in dorsal view, about 45 mm in length. b Specimen MZUSP PL syntype NHM B 2775 from sagittal sections. (The high version of this 519 in dorsal view. about 40 mm in length. c Diagrammatic figure can be found in the Electronic Supplementary Material) representation of the pharynx of specimen MZUSP PL 651 in lateral transverse fibers is present in the cephalic region as described columnar, ciliated epithelium. This anterior half is crossed by elsewhere (Carbayo and Leal-Zanchet 2003). numerous glands secreting erythrophil granules (1–2 μmin Mouth located in the middle of the pharyngeal pouch; this diameter), and surrounded by a 50-μm-thick circular muscle pouch at a maximum distance from penis bulb ranging from 0.0 layer. Distal half of the vesicle irregular in shape, either ex- to 2.5 mm (Fig. 3c). Pharynx bell-shaped (all specimens from panded dorso-ventrally or latero-laterally and broadly com- São Francisco de Paula) or nearly collar-shaped (one syntype— municated with the male atrium; this distal half of the vesicle from Taquara—and the specimen from Parobé), having the is lined by a ciliated epithelium which is very richly pierced by dorsal insertion considerably behind mouth level. Outer pha- glands producing gross xanthophil granules (2–3 μmindiam- ryngeal musculature consisting of a thin (3 μm) subepithelial eter), some glands producing erythrophil granules (<1 μmin longitudinal muscle, followed by a layer (40 μm) of diameter), and also by cells secreting dark-pinkish or intermingled circular and longitudinal muscles. Inner pharyn- cyanophil granules, the latter only distally. This distal half of geal musculature consisting of a circular muscle layer (50 μm the vesicle is surrounded by scarce muscle fibers apparently thick) with interspersed longitudinal fibers (MZUSP PL 1162). longitudinal. There is no ejaculatory duct, nor a penis papilla. Testes dorsal, located under supraintestinal transverse mus- Male atrium elongate, as long as 1.6–2.9 times that of fe- cle layer. They are arranged in two rows on each side of the male atrium (n = 8). The male atrium is provided with trans- body, each side with two to four testes in the same transverse verse folds filling its lumen. Folds are larger distally; the larg- plane. They extend from the level of the ovaries to 300 μm est one is dorsal to the gonopore canal, and separates the male before root of pharynx. Sperm ducts dilated distally by mass atrium from the female (Figs. 3dand4a). Male atrium prox- of sperm. Near prostatic vesicle, they curve medially, and imally lined by an 8-μm-high epithelium and with basal nuclei penetrate the ventro-lateral aspect of the common muscle coat. that reach 12–15 μm high distally. This epithelium is pierced Subsequently, either they communicate with each other before by abundant glands producing xanthophil granules (<1 μm) opening into the most proximal portion of the prostatic vesicle and scarce cells producing finer erythrophil granules, and is or this portion receives them separately (Figs. 3dand4a). surrounded by a circular muscle layer (5 μ m thick), which is Anterior half of prostatic vesicle (Figs. 3dand4a–c) cana- followed by a well-developed 100–150-μm-thick longitudinal licular, and oriented dorsally and posteriorly; it is lined by a layer of muscle fibers. Rolling into the deep of the land planarian genus Choeradoplana (Tricladida, Continenticola, Geoplanidae)... 197

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Fig. 4 Choeradoplana iheringi Graff 1899. a Diagrammatic of a sagittal section of the prostatic vesicle of specimen MZUSP PL 512. representation of the copulatory apparatus of specimen MZUSP PL 651 (The high version of this figure can be found in the Electronic from sagittal sections. b Photomicrograph of a sagittal section of the Supplementary Material) copulatory apparatus of specimen MZUSP PL 512. c Photomicrograph

Ovaries ovoid, 700 μm in longitudinal body axis, and Material studied Type material collected in Parque Nacional 200 μm in transverse plane. They are placed above the da Serra da Bocaina, Salesópolis, São José do Barreiro (São ventral nerve plate, and at a distance from anterior and Paulo, Brazil), approximately −22.74, −44.61, by F. Carbayo equal to 27–36% of body length. Ovovitelline ducts et al. Holotype MZUSP PL 541 (field number F2844): emerge from dorso-external aspect of ovaries. Laterally August 2008, transverse sections of cephalic region on 34 to female atrium, they rise posteriorly and medially, then slides, horizontal sections of ovaries region on 19 slides, trans- they unite dorsally to the common glandular ovovitelline verse sections of pre-pharynx on 15 slides, sagittal sections of duct (Figs. 3dand4a). They receive shell glands into their pharynx on 22 slides and sagittal sections of copulatory appa- distal portion. ratus on 50 slides, anterior and posterior end in 80% ethanol. Common glandular ovovitelline duct posteriorly and Paratype MZUSP PL 0540 (field number F2840): 11 downward directed; it is continuous with the female genital September 2008, sagittal sections of copulatory apparatus on duct. This duct runs postero-dorsally from the posterior por- 41 slides, anterior and posterior portions in 80% ethanol. tion of female atrium. Anterior portion of this duct ciliated and Paratype MZUSP PL 1158 (field number F2847): 11 pierced by glands producing cyanophil secretion; posterior September 2008, horizontal sections of ovaries region on 8 portion with stratified aspect, and pierced by two types of slides, transverse sections of pre-pharynx on 3 slides and sag- glands, producing cyanophil secretion and erythrophil gran- ittal sections of pharynx and copulatory apparatus on 10 ules, respectively. slides, anterior end in 100% ethanol. Additional specimens. Female atrium irregular in shape, with folded wall SMF N108 (Senckenberg Museum Frankfurt): Ribeirão Pires projecting into its lumen (Figs. 3dand4a). This atrium is lined (State of São Paulo), Coll. E. Bresslau, 10 November 1913, by a 20–30-μm-high columnar epithelium, and is crossed by sagittal sections of pharynx and copulatory apparatus on 6 two types of glands, producing cyanophil secretion and slides. SMF N271: Teresópolis (State of Rio de Janeiro), erythrophil granules, respectively. This epithelium is Coll. E. Bresslau, 28 May 1929, sagittal sections of pharynx surrounded by 10–15-μm-thick muscle layer of mainly longi- and copulatory apparatus on 6 slides. Aspecimennot tudinal muscles. They are continuous with muscle fibers of the labeled, described by Marcus (1951): Ribeirão Pires (State stroma, which are variously oriented. of São Paulo), sagittal sections of pharynx on one slide, sag- ittal sections of copulatory apparatus on two slides.

Choeradoplana pucupucu sp. n. Etymology In (indigenous Brazilian tribe), pucupucu means very long (Tibiriçá 1984). It refers to the Choeradoplana iheringi:Riester(1938), in part; Marcus relatively long prostatic vesicle. (1951); Leal-Zanchet and de Souza (2003), in part. Choeradoplana iheringi sensu Leal-Zanchet and Souza: Type locality Parque Nacional da Serra da Bocaina, São José Carbayo et al. (2013). do Barreiro, São Paulo, Southeast Brazil. 198 Carbayo F. et al.

Diagnosis Species of Choeradoplana with dorsal color pattern glandular cushions typical of the genus, is facing up (Fig. 5a, mottled with brownish speckles. CMI, 23.6–27.7%. Sperm b); posterior end pointed. ducts penetrate antero-lateral wall of penis bulb. Prostatic ves- Marcus (1951) described the general aspect of the dorsum icle intrabulbar, with proximal half canalicular, sinuous, and as black, mottled with clear areas, and a light gray ventral side. oriented posteriorly. Copulatory apparatus relatively long. No The worms we examined show a dorsum with general aspect permanent penis papilla. Male atrium lined with a ciliated light-to-dark brown produced by the cream-to-orange brown epithelium, which is underlain by a sponge-like tissue. Male ground color which is covered with numerous olive brown atrium elongate. Male:female atrial length ratio, 1.0–1.3. speckles. Ventral surface oyster white, with the exception of the brownish glandular cushions. Creeping sole as wide as Molecular diagnosis This species includes all populations 85% of body width. Mouth positioned at a distance from an- that cluster with individuals MZUSP PL 540–541 and terior end equal to 52–64% of body length; gonopore, at 74– MZUSP PL 1158 (Table 1) from this study, with significant 85%. Eyes formed by one pigmented cup with 40–80 μmin support in an adequate molecular delimitation model. diameter. No apparent clear halos around them. Eyes absent in the first 2 mm of the body, the equivalent of 4% of the body Geographic distribution and habitat Municipalities of length. Posteriorly, they are irregularly arranged in a marginal Teresópolis (State of Rio de Janeiro, Brazil), São José do row of 2–6 eyes throughout the body length. Barreiro, Salesópolis, and Ribeirão Pires (State of São Paulo, Sensory pits, 10 μm deep, distributed ventro-laterally in a Brazil). The species was found in areas covered with Atlantic uniserial row initiating 2 mm behind the anterior end, the forest. equivalent of 4.3% of body length. At the pre-pharyngeal region, most conspicuous glands are rhabditogenic, which pierce abundantly dorsal and marginal epidermis. This type Description of cells is scarce on the ventral epidermis; instead, erythrophil glands are more abundant. Glandular margin absent. Ventral Living adult specimens measured up to 50 mm in length and nerve plate with 80 μminthickness. 3.5 mm in width when creeping. Body slender, subcylindrical Cutaneous musculature as in the type species of the genus in cross section. Cephalic region slightly expanded laterally in terms of organization. In the cephalic region, cutaneous and rolled up so that ventral surface, provided with the two muscle organized and developed as in the type species of the

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Fig. 5 Choeradoplana pucupucu sp. n. holotype. a Creeping, in dorsal view, about 45 mm in length. b Creeping, in lateral view. c Photomicrograph of a sagittal section of the pharynx. (The high version of this figure can be found in the Electronic Supplementary Material) Rolling into the deep of the land planarian genus Choeradoplana (Tricladida, Continenticola, Geoplanidae)... 199 genus. In the pre-pharyngeal region, the longitudinal layer is 26% of body length. Ovovitelline ducts emerge from dorso- 130 μm thick dorsally, and is composed of dense bundles of external aspect of ovaries. Laterally to female atrium, they rise 60–75 fibers each. Ventrally, this layer is 90 μminthickness posteriorly and medially, then they unite dorsally to the and is composed of loose bundles with 8–12 fibers each. CMI, posteriormost portion of the female atrium (Fig. 6a). They 23.6–27.7%. Parenchymal musculature as in the type species receive shell glands into their distal portion. of the genus in terms of organization and relative thickness. Common glandular ovovitelline duct posteriorly and down- Mouth located in the end of anterior half of the pharyngeal ward directed; it is continuous with the female genital duct. This pouch; this pouch at 0.5 mm from penis bulb. Pharynx bell- duct runs postero-dorsally from the posterior portion of female shaped (Fig. 5c). Pharyngeal musculature as in the type spe- atrium, and is lined by a 20–35-μm-high epithelium and is cies of the genus in terms of organization and thickness. pierced by glands producing erythrophil granules. Testes located under the supraintestinal transverse muscle Female atrium long, with lateral folds filling its lumen. This layer. They are arranged in two rows on each side of the body, atrium is lined by a 20-μm-high columnar epithelium, the basal each side with three to five testes in the same transverse plane. portion of which is cyanophil. Ii is surrounded by a relatively They extend from the level of the ovaries to the root of phar- thick but loose circular muscle with longitudinal fibers inter- ynx. Sperm ducts dilated distally by mass of sperm. Laterally spersed. This layer is continuous with the thick muscle coat to the prostatic vesicle, they curve medianly to penetrate the composed by circular muscle fibers interspersed with longitu- lateral wall of the common muscle coat (Fig. 6a). dinal fibers. The female atrium receives erythrophil and Subsequently, the ducts run anteriorly and medianly to open cyanophil granules, each produced by a type of gland. into the very proximal portion of the prostatic vesicle. Proximal third of prostatic vesicle canalicular and very sin- Choeradoplana abaiba sp. n. uous, running posteriorly (Fig. 6a–c). This proximal third is lined by a columnar, ciliated epithelium which is pierced by Material studied Type material collected by F. Carbayo et al. two types of glands producing abundant erythrophil fine gran- in Parque Estadual da Serra do Tabuleiro (Santa Catarina, ules, and scattered cyanophil fine granules, respectively Brazil). Holotype MZUSP PL 1166 (field number F3864): (Fig. 6c). This anterior third is surrounded by a thin circular São Bonifácio (Santa Catarina, Brazil), approximately muscle (10 μm). Distal two thirds of the prostatic vesicle −27.84, −48.92, 12 July 2009, horizontal sections of cephalic gradually more expanded, with folded walls, and broadly region on 35 slides, transverse sections of post region of the communicated with the male atrium. The two distal thirds of ovaries on 9 slides, sagittal sections of pharynx on 16 slides, the prostatic vesicle are lined by an epithelium, which is irreg- sagittal sections of copulatory apparatus on 22 slides, posterior ularly ciliated, and pierced by the same two types of glands as end in 80% ethanol. Paratype MZUSP PL 509 (field number those of the canalicular portion; however, the very distal sec- F3312): São Bonifácio, −27.838333, −48.922500, 17 January tion of the prostatic vesicle, convergent with the epithelium of 2009,transversesectionsofcephalicregionon14slides,hor- the male atrium, is pierced by very abundant glands producing izontal sections of ovaries region on 14 slides, horizontal sec- coarse intensely xanthophil granules (gl in Fig. 6a–c). A few tions of post region of the ovaries on 16 slides, transverse circular muscle fibers surround the epithelium of this very sections of pre-pharynx on 15 slides, sagittal sections of phar- distal section. A penis papilla and ejaculatory ducts are absent. ynx on 15 slides, sagittal sections of copulatory apparatus on Male atrium elongate, as long as 1–1.3 times that of female 13 slides, posterior end in 80% ethanol. Paratype MZUSP atrium (n = 4). This male atrium presents folded walls occu- PL 503 (almost fully mature, field number F3240): Paulo pying its lumen; the largest fold projects backward from its Lopes, −27.947500, −48.751111, 12 January 2009, horizontal ventral distal half, eventually into the female atrium (Fig. 6a, sections of a portion including ovaries on 30 slides, transverse b). Male atrium lined by a 12-μm-high ciliated epithelium; sections of pre-pharyngeal region on 12 slides, sagittal sec- cilia are shorter on the large fold. The epithelium is tions of pharynx on 27 slides, sagittal sections of copulatory surrounded by a 5-μm-thick circular muscle, followed by a apparatus on 44 slides, posterior end in 80% ethanol. 2–5 times thicker longitudinal muscle, and is pierced by Paratype MZUSP PL 595 (field number F3221), not mature: glands producing erythrophil granules. Under epithelium of Paulo Lopes, −27.961389, −48.758333, 11 January 2009, the large fold, circular muscle fibers are thinner and embedded transverse sections of cephalic region on 16 slides, horizontal in a weakly cyanophil mass of undefined limits resembling the sections of a portion including ovaries on 22 slides, transverse sponge-like tissue found beneath the epithelium of the inverted sections of post region of the ovaries on 12 slides, sagittal penis of C. minima (Lemos et al. 2014)(Fig.6d). Stroma of the sections of pharynx on 11 slides, posterior end in 80% ethanol. large fold is constituted by numerous circular muscles. Ovaries elongate, with 700 μm in longitudinal body axis, Etymology In Tupi language, abaíba means difficult, arduous and 100 μm in transverse plane. They are placed above the (Tibiriçá 1984). It refers to the difficulty in delimiting the ventral nerve plate, and at a distance from anterior end equal to taxonomic level of its morphological and molecular variation. 200 Carbayo F. et al.

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Fig. 6 Choeradoplana pucupucu sp. n. holotype. a Diagrammatic sagittal section of the prostatic vesicle. d Photomicrograph of a sagittal representation of the copulatory apparatus. b Photomicrograph of a section of large fold of distal portion of the male atrium. (The high version sagittal section of the copulatory apparatus. c Photomicrograph of a of this figure can be found in the Electronic Supplementary Material)

Type locality Parque Estadual da Serra do Tabuleiro, State of and MZUSP PL 1166–70 (Table 1)fromthisstudy,withsig- Santa Catarina, South Brazil. nificant support in an adequate molecular delimitation model.

Diagnosis Species of Choeradoplana with dorsal color pat- tern mottled with brownish speckles. CMI, 27–28%. Sperm Geographic distribution and habitat The species was only ducts penetrate antero-ventral or antero-lateral wall of the pe- found in the type locality, which is covered by Atlantic forest. nis bulb. Prostatic vesicle intrabulbar, its proximal half is a canal oriented dorsally or nearly horizontally. No permanent penis papilla. Male:female atrial length ratio, 2.7–5.0. Description

Molecular diagnosis This species includes all populations Living adult specimens not measured. Fixed, up to 47 mm in that cluster with individuals MZUSP PL 503, MZUSP PL length and 4.5 mm in width. Body slender; flattened at rest 505, MZUSP PL 509, MZUSP PL 511, MZUSP PL 595, (Fig. 7a). Cephalic region slightly expanded laterally and rolled Rolling into the deep of the land planarian genus Choeradoplana (Tricladida, Continenticola, Geoplanidae)... 201

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c d

e f

Fig. 7 Choeradoplana abaiba sp. n. a Holotype, in dorsal view, about Photomicrograph of a sagittal section of the copulatory apparatus of the 40 mm in length. b Photomicrograph of a sagittal section of the pharynx holotype. f Diagrammatic representation of the sperm ducts and prostatic of specimen MZUSP PL 509. c Diagrammatic representation of the vesicle of the specimen MZUSP PL 503. (The high version of this figure copulatory apparatus of the holotype. d Photomicrograph of a sagittal can be found in the Electronic Supplementary Material) section of the prostatic vesicle of the specimen MZUSP PL 503. e up so that the ventral surface, provided with glandular cushions the equivalent of 3–5% of the body length. Posteriorly, they are typical of the genus, is facing up; posterior end pointed. Ground irregularly arranged in a marginal row of 3–8 eyes throughout color of the body ochre yellow. In the East population (Paulo the body length. Sensory pits, 10 μm deep, distributed ventro- Lopes), pigment is densely distributed excepting for irregular or laterally in a uniserial row initiating 1–2mmbehindtheanterior rounded clear areas and a tenuous midline in anterior two thirds. end, the equivalent of 1.6–4.2% of the body length. At the pre- In the West population (São Bonifácio), dorsum covered with pharyngeal region, most conspicuous glands are rhabditogenic, brown speckles forming a reticulated ornament; speckles dense- which pierce abundantly dorsal and marginal epidermis. This ly distributed medianly, and a loosely marginally. 1/5th posterior type of cell is scarce on the ventral epidermis; instead, darker. Ventral surface sand yellow, with the exception of the erythrophil glands are relatively abundant. Glandular margin orange-brown glandular cushions. Creeping sole as wide as 76– absent. Ventral nerve plate with 70–80 μm in thickness. 81% of body width. Mouth positioned at a distance from ante- Position of cerebral ganglia not determined. rior end equal to 50–60%; gonopore, at 65–80%. Eyes formed Cutaneous musculature organized as in the type species of by one pigmented cup with 45–50 μmindiameter.Noclear the genus. Dorsally, the longitudinal layer is 145 μminthick- halos around them. Eyes absent in anterior 1.5–2.5 mm of body, ness and is composed of dense bundles of 40–140 fibers each. 202 Carbayo F. et al.

Ventrally, this layer is 150–175 μm in thickness and is com- longitudinal and circular fibers interspersed. This layer is bet- posed of loose bundles with 10–40 fibers each. CMI, 27–28%. ter delimited in West population. Parenchymal musculature as in the type species of the genus Ovaries pear-shaped, 400 μm in longitudinal length, and in terms of organization and relative thickness. 200 μm in transverse plane. They are placed above the ventral Mouth located shortly anterior to middle of the pharyngeal nerve plate, and at a distance from anterior end equal to 28– pouch; this pouch at 0.4–1.8 mm from penis bulb. Pharynx 30% of body length. Ovovitelline ducts emerge from dorso- bell-shaped, having the dorsal insertion behind mouth level external aspect of ovaries. Laterally to female atrium, they rise (Fig. 7b). Pharyngeal musculature as in the type species of posteriorly and medially; subsequently, they unite above pos- the genus in terms of organization and thickness. terior section of female atrium (Fig. 7c). They receive shell Testes dorsal, located under supraintestinal transverse glands into their distal portion. Common glandular muscle layer. They are arranged in two rows on each side ovovitelline duct directed posteriorly and ventrally; it is con- of the body, each side with one to three testes in the same tinuous with the female genital duct. In fully mature speci- transverse plane. They extend approximately from the level mens, this duct runs postero-dorsally from the posterior por- of ovaries (from 2.5 mm posterior to the level of ovaries in tion of female atrium; the epithelium of the portion converging one specimen from West population) to 0.5 mm before root to the common duct is ciliated and 10–20 μm high. Then, it of pharynx. Sperm ducts dilated distally by mass of sperm. increases in height to 30 μm, and its free surface becomes Anterior portion of male organ is variable. In West popu- irregular, and pierced by glands producing cyanophil secre- lation, sperm ducts penetrate the ventro-lateral wall of the tion, and by a second type, scarce, producing fine erythrophil common muscle coat (Fig. 7c, e), whereas in East popula- granules. tion, they penetrate the wall laterally (Fig. 7d, f). In fully mature specimens, female atrium roughly funnel- Subsequently, they run anteriorly and open into the very shaped, with two to three dorsal and posterior folds (Fig. 7c). proximal portion of prostatic vesicle (Fig. 7c) or into the This female atrium is lined by a 20–30-μm-high columnar lateral aspect of the proximal portion (Fig. 7f). epithelium, the free surface of which is irregular in some sec- Anterior half of prostatic vesicle is either a horizontal, tions. This epithelium is crossed by three types of glands, one canalicular, and with slightly folded walls (East population; producing fine erythrophil granules, a second producing Fig. 7d, f) or a canal dorso-anteriorly inclined, and with cyanophil granules, and a third one producing very abundant richly folded wall (West population) (Fig. 7c, e). This an- amorphous and weakly cyanophil secretion. The epithelium terior half is lined by a columnar, 25-μm-high ciliated ep- of this atrium is surrounded by a single longitudinal muscle ithelium which is crossed by glands secreting fine layer followed by a single circular layer. Space between this erythrophil granules (0.5 μm in diameter), and surrounded double layer and the common muscle coat is consistently oc- by a 25-μm-thick circular muscle layer. Posterior half of cupied by the glands producing weakly cyanophil secretion. the prostatic vesicle either expanded latero-laterally or dorso-ventrally. This half is lined by a ciliated epithelium, Choeradoplana agua sp. n. which is very richly pierced by glands producing fine xanthophil granules (<0.5 μm in diameter), by less abun- Material studied Type material collected in Santa Maria dant glands producing erythrophil granules (<1 μmindi- Madalena (Rio de Janeiro, Brazil), −21.876667, −41.919722 ameter), and by cells the thick neck of which is 5–7 μmin by J. Pedroni et al. Holotype MZUSP PL 2059 (field number diameter and produce amorphous cyanophil secretion; this F3967): 9 August 2009, horizontal sections including ovaries distal half of prostatic vesicle surrounded by circular mus- on 12 slides, transverse sections of pre-pharyngeal on 6 slides, cle layer, 5–10 μmthick,followedbya20–35-μm-thick sagittal sections of pharynx and copulatory apparatus on 68 layer of longitudinal fibers with circular fibers interspersed. slides, anterior and posterior portions in 80% ethanol. Male atrium elongate, as long as 2.7–5 times that of female. Paratypes: MZUSP PL 446 (incompletely mature, field This male atrium is provided with large transverse folds filling number F2205): 18 March 2008, transverse sections of ce- its lumen; distal fold is dorsal to gonopore canal, and separates phalic region on 14 slides and sagittal sections of pharynx the male atrium from the female (Fig. 7c). The male atrium is and copulatory apparatus on 23 slides, remaining body in lined by an epithelium, 8–10 μm high proximally, and 25 μm 80% ethanol. MZUSP PL 445 (incompletely mature, field high distally; anterior half pierced by abundant glands produc- number F2204): 18 March 2008, horizontal sections of a por- ing xanthophil granules (<1 μm in diameter), and by scarce tion behind anterior end on 8 slides, transverse sections of pre- and irregularly distributed glands with necks 7 μmindiameter pharyngeal region on 8 slides, sagittal sections of copulatory that produce amorphous weakly cyanophil secretion; distal apparatus on 13 slides, posterior end in 80% ethanol. MZUSP half pierced by the latter type of gland and also by abundant PL 619 (field number F3952): 9 August 2009, transverse cells producing fine erythrophil granules. Atrial epithelium is sections of cephalic region on 9 slides, horizontal sections of underlined by a 100–150-μm-thick muscular layer of ovaries region on 29 slides, transverse sections of post region Rolling into the deep of the land planarian genus Choeradoplana (Tricladida, Continenticola, Geoplanidae)... 203 of the ovaries on 8 slides and sagittal sections of pharynx and gonopore, at 71–78.1%. Eyes formed by one pigmented cup, copulatory apparatus on 34 slides, posterior end in 80% 40–45 μm in diameter. No clear halos around them. Eyes absent ethanol. in the first 3 mm of the body, the equivalent of 8% of the body length. Posteriorly, they are irregularly arranged in a marginal Etymology In Tupi language, aguá means bubble and row of 2–6 eyes throughout the body length. ampoule means swelling (Tibiriçá 1984). It refers to the shape Sensory pits, 10 μm deep, distributed ventro-laterally in a of the prostatic vesicle. uniserial row initiating 0.4 mm behind the anterior end, the equivalent of 0.8% of body length. At the pre-pharyngeal region, Type locality Parque Estadual do Desengano, Santa Maria most abundant glands are rhabditogenic, which pierce dorsal and Madalena, State of Rio de Janeiro, Southeast Brazil. marginal epidermis. This type of cells is scarce on the ventral epidermis; instead, erythrophil glands are more abundant. Diagnosis Species of Choeradoplana with dorsal color pat- Glandular margin absent. Ventral nerve plate with 140 μmin tern mottled with brownish spots. CMI, 33.7–34.8%. thickness; location of cerebral ganglia not determined. Proximal portion of prostatic vesicle extrabulbar, tubular, The cutaneous musculature comprises three layers arranged and initially bifurcated; intrabulbar portion pear-shaped with throughout the body as in the type species of the genus. CMI, intensely folded wall. No permanent penis papilla. 33.7–34.8%. Parenchymal musculature as in the type species of Male:female atrial length ratio, 4–6. the genus in terms of organization and relative thickness. Mouth located in the middle of the pharyngeal pouch; this Molecular diagnosis This species includes all populations pouch at 1.4 mm from copulatory apparatus. Pharynx cylin- that cluster with individuals MZUSP PL 445–46, MZUSP drical to bell-shaped, having the dorsal insertion approximate- PL 448, and MZUSP PL 619 (Table 1) from this study, with ly at the mouth level (Fig. 8b). Pharyngeal musculature as in significant support in an adequate molecular delimitation the type species of the genus in terms of organization and model. relative thickness. Testes dorsal, located under supraintestinal transverse mus- Geographic distribution and habitat The species was only cle layer. They are arranged in two rows on each side of the found in Atlantic forest in the type locality. body, each side with one to three testes in the same transverse plane. They extend from the level of the ovaries to the root of pharynx. Sperm ducts dilated distally by mass of sperm. Description Beneath prostatic vesicle, they curve medianly and open into the proximal, short branched portion of the prostatic vesicle. Living adult specimens measured up to 60 mm in length and The branches of the vesicle join each other and then are con- 4 mm in width. Body slender, subcylindrical in cross section. tinued forward and dorsally as an unpaired portion. This un- Cephalic region slightly expanded laterally and rolled up so that paired portion penetrates the ventral aspect of the common ventral surface, provided with the two glandular cushions typical muscle coat to communicate with the intrabulbar portion of the genus, is facing up; posterior end pointed (Fig. 8a). (Fig. 9a). The intrabulbar portion of the prostatic vesicle is a Ground color of the dorsum beige, covered with clay brown bulb-shaped structure dorso-posteriorly inclined; its walls are pigment spots; these spots, free or anastomosed; at the level of intensely projected into its lumen (Fig. 9a–c). The prostatic the copulatory apparatus, spots more densely distributed; on vesicle communicates with the antero-dorsal portion of the body margins, spots scattered. Ventral surface sand yellow, with male atrium. An ejaculatory duct and a penis papilla are absent. the exception of the orange-brown glandular cushions. Creeping The epithelium of the vesicle ranges from 25 to 40 μmin sole as wide as 90% of body width. Mouth positioned at a height. Cilia are only present on the epithelium lining the paired distance from anterior end equal to 63.1–64% of body length; portion and that of the very distal one that converges with the

Fig. 8 Choeradoplana agua sp. ab n. (a) Paratype MZUP PL 619 in dorsal view, about 50 mm in length. b Diagrammatic representation of the pharynx of the holotype. (The high version of this figure can be found in the Electronic Supplementary Material) 204 Carbayo F. et al.

a

bc d

Fig. 9 Choeradoplana agua sp. n. holotype. a Diagrammatic Photomicrograph of a sagittal section of the copulatory apparatus at the representation of the copulatory apparatus. b Photomicrograph of a level of the gonopore canal. (The high version of this figure can be found sagittal section of the copulatory apparatus. c Photomicrograph of a in the Electronic Supplementary Material) sagittal section of the intrabulbar portion of prostatic vesicle. male atrium. Regarding the secretions, the vesicle is divided portion present its surface erythrophil, but apparently is not into several sections: the paired portion is pierced by two types pierced by glands (Fig. 9a–c). The prostatic vesicle is of glands producing pale erythrophil and cyanophil granules, surrounded by a 50-μm-thick muscle layer of crisscrossed fi- respectively; the unpaired, extrabulbar portion is pierced by the bers, excepting distally, where fibers are longitudinal. same two types of glands, but they are more intensely stained. Male atrium elongate, as long as 4–6 times that of female These glands gradually are replaced by two types of conspicu- atrium length (Fig. 9a). This male atrium presents a few large ous glands in most of the intrabulbar, bulb-shaped portion (g1 folds occupying its lumen. The largest fold is ring-shaped and in Fig. 9c), namely, very numerous glands producing gross located distally in a transverse plane. The anterior two thirds erythrophil granules, and less abundant glands secreting fine of the male atrium are lined by a 6-μm-high epithelium which cyanophil granules, then follows a portion (g2) pierced by is surrounded by a thin circular muscle followed by a thick abundant glands producing gross cyanophil granules and by (10 μm) longitudinal muscle. The distal third is lined by a glands secreting fine erythrophil granules. A short section 25-μm-high columnar epithelial cells, the apical portion of (g3) close to the distal region of the vesicle is pierced by glands which cumulate xanthophil granules (Fig. 9d). A 10-μm- secreting erythrophil granules. The very distal (g4), ciliated thick muscle of crisscrossed fibers surrounds this distal Rolling into the deep of the land planarian genus Choeradoplana (Tricladida, Continenticola, Geoplanidae)... 205 portion of the male atrium. Stroma of large fold is constituted Concerning the internal organs, nine of the 15 species by numerous circular muscles and scarce longitudinal (C. ehrenreichi is here not considered as its internal organs muscles. are unknown) stand apart from the four species described here; Ovaries ovoid, 500 μm in longitudinal body axis; and 300 μm in that, they present at least one of these conspicuous, distinc- in transverse plane. They are placed above the ventral nerve plate, tive features: dorsal and ventral cutaneous longitudinal muscle and at a distance from anterior end equal to 44% of body length. partially insunk into the parenchyma (C. gladismariae), a com- Ovovitelline ducts emerge from dorso-external aspect of ovaries. pact copulatory apparatus (C. langi Graff, 1894 (Bois- Laterally to female atrium, they rise posteriorly and medially, then Reymond 1951), C. marthae, C. crassiphalla), a permanent anteriorly, and subsequently unite behind the female atrium penis papilla (C. crassiphalla, C. benyai, C. marthae), an (Fig. 9a). They receive shell glands into their distal portion. inverted penis (C. minima), a prostatic vesicle intensely pleated Common glandular ovovitelline duct downward directed; in its distal portion (C. catua, C. gladismariae), a massive it is continuous with the female genital duct. This duct runs sphincter ventral to male and female atria (C. banga), a female postero-dorsally from the posterior portion of female atrium; it genital atrium tubular (C. langi, C. bilix), and a female genital is lined by a 12-μm-high epithelium and is pierced by cells canal approaching female atrium from the ventral side producing erythrophil granules. (C. banga) (Carbayo and Froehlich 2012; Bois-Reymond Female atrium short and ample, with lateral folds filling its 1951; Froehlich 1955b;Graff1899; Lemos et al. 2014; lumen (Fig. 9a). The lining epithelium, as well as its surround- Negrete and Brusa 2012). ing muscle, and the glands piercing it are similar to those The remaining six species can be grouped according to the lining the ring-shaped fold of the male atrium (Fig. 9d). location of the prostatic vesicle, this being either extrabulbar in three species (Choeradoplana agua sp. n., C. albonigra, and C. bocaina) or intrabulbar in another three species (C. iheringi, C. pucupucu sp. n., and C. abaiba sp. n.) Comparative discussion of the described species (Carbayo and Froehlich 2012;Graff1899;Riester1938). The species presenting an intrabulbar prostatic vesicle dis- The species (re)described in this work can be molecularly and tinguish readily from each other as regards of the shape of this morphologically differentiated from each other and from the structure. In C. agua sp. n., the prostatic vesicle is very distinct remaining congeners. Regarding the external aspect, two dorsal in having its proximal, extrabulbar portion initially bifurcate color appearances can be recognized in Choeradoplana, namely, and tubular, and the intrabulbar is portion pear-shaped with a longitudinally striped pattern and a mottled pattern. The striped intensely folded wall. In C. benyai, the extrabulbar portion is pattern is found in all representatives of the genus with the ex- dilated, whereas in C. bocaina, it is dish-shaped. Furthermore, ception of the four species morphologically studied here plus the male atrium of C. agua sp. n. is richly folded, while in C. bocaina, C. benyai, C. crassiphalla,andC. banga (Carbayo C. albonigra and C. bocaina presents large folds (Carbayo and Froehlich 2012; Lemos et al. 2014; Negrete and Brusa and Froehlich 2012; Lemos et al. 2014; Riester 1938). 2012). These eight species present a general pattern consisting C. iheringi and C. pucupucu sp. n. are both taxonomic of brownish speckles or dots on a light-brown ground color. entities that have been considered conspecific for a long time Choeradoplana banga, C. benyai, C. crassiphalla, C. iheringi, (see Graff 1899;Riester1938;Marcus1951;Froehlich1955b; C. abaiba sp. n., and C. pucupucu sp. n. confuse with each other Froehlich 1956; Leal-Zanchet and de Souza 2003). These two because they present brownish speckles very similar in color and species and C. abaiba sp. n. present an intrabulbar prostatic distribution (Carbayo and Froehlich 2012;Graff1899;Lemos vesicle, but only in C. pucupucu sp. n., (i) the proximal half of et al. 2014; Negrete and Brusa 2012), whereas C. bocaina and the prostatic vesicle is a long and sinuous canal; (ii) the male C. agua sp. n., both ornate with brownish spots, are mostly alike atrium is lined with a ciliated epithelium, which is underlain to each other (Carbayo and Froehlich 2012). Therefore, conclu- by a sponge-like tissue (like that found in C. minima); and (iii) sive identification of the species here covered is not possible by the female atrium is as long as the male one. Furthermore, in means of the external aspect. An exception might be the popu- C. pucupucu sp. n., the sperm ducts penetrate wall of the penis lation of C. pucupucu sp. n. from Ribeirão Pires; the dorsum of bulb antero-laterally, whereas in C. iheringi entrance is antero- specimens from this locality is black, mottled with clear areas ventrally. In C. abaiba sp. n., entrance is either the antero- (Marcus 1951) and, therefore, can be distinguished from the ventrally (West population) or antero-laterally (East). other species having the mottled pattern. In this regard, C. iheringi and C. abaiba sp. n., on their part, are very alike. conspecificity of this population could be doubt, but variation They differ from each other; in that, in C. iheringi, (a) the in the dorsal chromatic pattern is well known in geoplaninids proximal portion of the prostatic vesicle is an approximately [e.g., C. bergi (Graff, 1899) in Marcus 1951; nungara straight canal oriented dorsally, while in C. abaiba sp. n., this Carbayo et al., 2016]; thus, this feature in itself is not reliable for portion is either a horizontal, canalicular with slightly folded species identification. wall (East population) or a canal dorso-anteriorly inclined with 206 Carbayo F. et al. richly folded wall (West population); (b) the male:female atrial are now well recognized as distinct species on the basis of length ratio is 1.6–2.9, whereas this ratio is 2.7–5.0 in C. abaiba internal traits and the molecular data, as is also the case of the sp. n. (and only 1.0–1.3 in C. pucupucu sp. n.); and (c) the new species C. agua sp.n.However,C. iheringi and C. abaiba dorsal insertion of the pharynx is strongly posterior to the ven- sp. n., which constitute a robust monophyletic clade in all mo- tral one, whereas in C. abaiba sp.n.(andC. pucupucu sp. n.), it lecular analyses, are also morphologically closest to each other is not so posteriorly displaced. and greatly similar (see comparative discussion of the described species). Indeed, without molecular support, morphological traits supporting their distinctiveness as real species would General discussion probably have been interpreted as intrapopulation variation. Another question is whether C. abaiba sp. n. may be divid- Molecular species delimitation performance ed into two species, as some of the delimitation methodologies indicated. We cannot overlook the different topologies found Cox1 by itself has shown to be quite good in delimiting species; in the phylogenies inferred only from the mitochondrial mark- however, some limitations exist. In the first place, depending on er Cox1 (Supp. Fig. 1) and from the information of the the methodology used for the delimitation, some differences concatenated (mitochondrial and nuclear) genes (Fig. 2). The arise (Fig. 2). These discrepancies based in the same dataset mitochondrial gene splits C. abaiba sp. n. into two distinct are clearly a consequence of the differences in the theoretical highly differentiated lineages paraphyletic with respect to bases of the methodologies used to do the delimitation. But, C. iheringi. The concatenated tree (Fig. 2)alsoshowsahigh what is more important here is the fact that the validation meth- difference between the two clades (C and D) in terms of ge- od used (BP&P), based in multilocus information and in the netic distance, although they are grouped in a monophyletic detection of gene flow, results in less species than those based clade. It is known that genetic data provide potential evidence exclusively on trees and distances, a fact that has already been of divergence at an early stage of diversification (Knowles and reported (Flot et al. 2011). In our case, the different assignations Carstens 2007). We observed slight morphological differences can be due both to the differences in the theoretical bases of the between the two populations of C. abaiba, which occur on the methodologies, but also on the fact that the BP&P method is East or West slopes of the Serra do Tabuleiro, respectively (see using multiple loci while the rest are based only in a mitochon- description and comparative discussion above). These differ- drial gene, which can result in the gene history not reflecting the ences might reflect an ongoing incipient speciation by isola- species histories. Moreover, mitochondrial genes that have tion. Accordingly, the mitochondrial genome would have al- higher rates of change and hence explain more shallow rela- ready begun to differentiate (showing a paraphyletic species in tionship can better detect initial steps of speciation but also will Cox1 phylogenies), while nuclear genes are still sharing infor- be more affected by incomplete lineage sorting (Schwarzfeld mation between populations similar to what has been reported and Sperling 2015; Batalha-Filho and Miyaki 2016). in other organisms, such as red deer (Carranza et al. 2016). Although it has recently been found that coalescent This situation reinforces the importance of analyzing multiple delimitation-based methods (as BP&P) may delimit genetic datasets, especially molecular information. These can show structure and not species (Sukumaran and Knowles 2017), this not only a frozen image of the present situation but also the is not our case. Using simultaneously this methodology and future direction of evolution for the lineages which is espe- morphology, we find that the results are fully coherent giving cially important in a conservation context. credibility to the coalescent and multigenic based delimitation, InthecaseofC. iheringi, according to the GMYC approx- and at the same time reinforcing the need of an integrative imation, the species is divided into two distinctive species (A protocol, as that followed here (and already in other publica- and B; Fig. 2 and Suppl. Fig. 1). However, we considered both tions: Carbayo et al. 2016;Mateosetal.2017), to have robust clades as a unique species based on the following consider- species delimitations and descriptions. ations: (a) the other three molecular delimitation methods coin- cide in considering A and B as a single species and (b) we failed Cryptic species under the guise of C. iheringi to detect traits underpinning morphological characterization of each clade. There is only a feature worthy to mention, namely, The most prominent finding from our integrative taxonomic the shape of the pharynx, either nearly collar-shaped—in spec- approach is the discovery of several cryptic species. By means imens from Taquara and Parobé—and bell-shaped—in speci- of their external aspect only, the four species here described mens from São Francisco de Paula. However, as both types of may have gone unnoticed. This is not an isolated case; there pharynx do occur in clade B, this trait does not delimit any are other well-documented examples of cryptic land planarians clade. In consequence, with the integrative criterion according (e.g., Ogren and Darlington 1991;Leal-Zanchetetal.2012; to which we consider, a different species must present both Carbayo and Almeida 2015; Álvarez-Presas and Riutort morphological and molecular differences; with the data at hand, 2014; Lemos et al. 2014). C. iheringi and C. pucupucu sp. n. the two clades belong to the same species. Rolling into the deep of the land planarian genus Choeradoplana (Tricladida, Continenticola, Geoplanidae)... 207

With regard to other species included in this study, our homogeneity. Two of the three species composing the analyses of molecular species delimitation show incongru- subclade A-F (C. iheringi and C. abaiba sp. n.) are notably ences for C. albonigra, C. bocaina,andC. benyai.For alike. However, their sister species, C. benyai,isverydiffer- C. albonigra, only ABGD and bPTP show two species, but ent; in that, it possesses a conspicuous, very large penis papil- the GMYC and the validation methodology, BP&P, coincide la, organ that is absent in C. iheringi and C. abaiba sp. n. Its with the morphological assignation, giving support to its spe- sister clade G-I is constituted by three species, among which cies status. However, in the case of C. bocaina and C. benyai, C. marthae is the only one having a dorsal color with a striped three out of the four molecular methods coincide in delimiting pattern and a copulatory apparatus very compact and, differ- two species (the discovery approaches), while the morpholog- ently from the other two members, also presents a penis pa- ical delimitation and BP&P concur in the classification of the pilla, which is not homologous with that of C. benyai two clades for each species as belonging to one single species. (Froehlich, 1955b; Lemos et al. 2014). The most basal species But here again, as in the case of C. abaiba sp. n., the genetic of the clade A-J is C. minima,fromSouthBrazil,whichis differentiation between the clades is high (both for Cox1 alone unique in the genus having an inverted penis. This type of and also for the concatenated dataset; Supp. Fig. 1 and Fig. 2). penis is known in a few Graff, 1896 These results highlight the possibility that these species also (Geoplanidae), such as Eudoxiatopoplana bilaticlavia hide incipient genetically independent lineages, still virtually Winsor, 2009, and species in Marionfyfea (Winsor, 2006, cryptic at the morphological level. In the case of C. bocaina, 2011) Interestingly, only Choeradoplana minima and in the original description, a paratype was noted to be some- C. pucupucu sp. n. present a sponge-like tissue underneath what different in external and internal aspects, in part due to the male atrium, but are not sister species. fixation artifacts (Carbayo and Froehlich 2012). Nonetheless, With respect to the second big clade K-O, the internal to properly test hypotheses of their conspecificity and disen- group L-N includes species also morphologically heteroge- tangle whether they hide cryptic species or not, an issue out of neous in the dorsal color pattern, being either mottled or the scope of this paper, additional material should be morpho- striped. Both patterns are found in the A-J clade, and conse- logically and molecularly studied. quently, the body color pattern is not considered homologous at the genus level. The outline of their copulatory apparatus is Phylogeny, morphology, and biogeography comparable, with highest similarity between C. albonigra and C. bocaina, which, however, are not sister species (Riester, The phylogeny obtained with the concatenated dataset (Fig. 2) 1938; Carbayo and Froehlich 2012). Choeradoplana included 12 of the 16 existing species of the genus gladismariae, the most basal species of the group, is unique Choeradoplana. Species relationships are highly supported, in the genus; in that, the cutaneous longitudinal muscles are yet only some subclades within each of the two big clades, partially sunken into the parenchyma, both dorsally and ven- namely, A-J and K-O, present some morphological trally (Carbayo and Froehlich 2012). This situation is

Fig. 10 Phylogenetic tree of Choeradoplana projected on the map (adapted from Fig. 2). Dashed lines indicate the lineages incongruentwithvicariance hypothesis. (The high version of this figure can be found in the Electronic Supplementary Material) 208 Carbayo F. et al. convergent with the other known case found in the Chilean suggestions. FC has financial support from FAPESP (proc. genus Froehlich, 1978. 2016/18295-5). M. R. and M. Á.-P. were supported by grant CGL2011- 23466 of the Ministerio de Economía y Competitividad, Spain. M. Á.-P. In sum, the uneven distribution of these morphological acknowledges support from SEG, the Sociedad Española de Genética traits (namely, body color, insunk of longitudinal cutaneous travel grant. muscle, general shape of copulatory apparatus, type of penis, sponge-like tissue underlaying the epithelium of the male atri- Abbreviations cm, common muscular coat; CMI, cutaneous musculature um) across Choeradoplana and Geoplanidae suggests conver- thickness relative to body height at the pre-pharyngeal region; co, com- gent evolution resulting in a lack of synapomorphies for spe- mon glandular ovovitelline duct; dd, dorso-diagonal parenchymal mus- cies groups. Morphological heterogeneity in internal groups cles; e,eye;ES, State of Espírito Santo, Brazil; es, esophagus; f,fold;fa, – of Choeradoplana could also be a consequence of an old female genital atrium; fg, female genital canal; g, gonopore; g1 g4,glan- dular sections of the prostatic vesicle; gl, glands; i, intestine; ls, normal radiation of the genus followed by a long period of differen- longitudinal cutaneous muscles; m, muscle fiber; ma, male genital atrium; tiation. This can also be conjectured from the long branches MG, State of Minas Gerais, Brazil; mo, mouth; MZU, Museu de Zoologia separating most species while the basal branches uniting them da Universidade do Vale do Rio dos Sinos; MZUSP, Museu de Zoologia are proportionally shorter, and from the fact that only the two da Universidade de São Paulo; ng, nervous ganglia; NHM,Natural History Museum Vienna; ov, ovoviteline duct; pg, sponge-like mass; species (C. iheringi and C. abaiba sp. n.) more derived and ph, pharyngeal pocket; PR, State of Paraná, Brazil; pv, prostatic vesicle; closely related to each other in the analyses still share mor- RJ, State of Rio de Janeiro, Brazil; RS, State of Rio Grande do Sul, Brazil; phological features. SC; State of Santa Catarina, Brazil; sd, sperm duct; sg, shell glands; sk, This scenario shows the unsuitability of these morpholog- sunken longitudinal cutaneous muscles; SMF, Senckenberg Museum Frankfurt; SP, State of São Paulo, Brazil; vi,vitellaria;vn, ventral nerve ical characters to infer evolutionary relationships and defining plate taxonomic lineages in contrast to the molecular data advan- tage to solve the phylogeny of the group. A similar situation was also proposed for the contrasting results between morpho- References logical (Ball 1981; Sluys 1989a) and molecular phylogenies of Tricladida (Carranza et al. 1998). Álvarez-Presas, M., & Riutort, M. (2014). Planarian (Platyhelminthes, The geographic distribution of the species reveals a general Tricladida) diversity and molecular markers: a new view of an old pattern after which the lineage represented by the big clade K- group. Diversity, 6,323–338. 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