Supporting Information Schärer et al. 10.1073/pnas.1013892108 SI Materials and Methods analyses, nodal support was estimated using ML bootstrapping PCR and Sequencing. For each species we sequenced (in one to (100 replicates) as implemented in GARLI version 0.942 (8) three specimens) the complete small subunit ribosomal DNA using default settings, except setting Genthreshfortopoterm to 4 (ssrDNA) and part (D1–D3) of the large subunit ribosomal DNA 10 generations. Clades were considered to have high nodal (lsrDNA) resulting in a total of ∼2,850 base pairs. Total genomic support if BI posterior probability was ≥95% and ML bootstrap DNA (gDNA) was extracted from the DNA samples using the resampling was ≥70%. DNeasy tissue kit (QIAGEN) following the manufacturer’s instructions; gDNA was eluted in 2× 100-μL volumes. PCR re- Constraint Analysis. We performed a constraint analysis that tested actions were carried out in 25-μL volumes using Illustra puReTaq whether the data support the a priori hypothesis of a single origin Ready-To-Go PCR beads (GE Healthcare), 1 μLof10μMof of the hypodermic mating syndrome. A constrained tree holding fi each primer (a list of primers is given in Table S2), and 1–2 μL the ve taxa with this feature as monophyletic was loaded as gDNA extract. Partial lsrDNA (1,142–1,189 base pairs) was am- a backbone constraint before ML analysis under the same model plified using ZX-1 (1) + 1500R (2); difficult templates were as the unconstrained tree (Fig. S2). Log likelihood scores of amplified with nested PCR using ZX-1 + 1200R and 300F + constrained and unconstrained trees were used in a Shimodaira– 3 1500R. ssrDNA (1,706–1,711 base pairs) was amplified using Hasegawa test (9), as implemented in PAUP*, with 10 RELL WormA + WormB; difficult templates were amplified with nested bootstrap replicates. PCR using Macro_18S_200F + Macro_18S_1640R. Cycling conditions for lsrDNA were as follows: denaturation for 5 min at Ancestral State Reconstruction. We performed ancestral state 95 °C followed by 40 cycles of 30 s at 95 °C, 30 s at 55 °C, 2 min at reconstructions to infer the character states at the base of the 72 °C, and 7 min extension at 72 °C. Cycling conditions for genus Macrostomum and the base of clade 2 (both nodes are well ssrDNA were as follows: denaturation for 2 min at 94 °C followed supported in the ML and BI analyses). We used Mesquite ver- by 40 cycles of 30 s at 94 °C, 30 s at 54 °C, 2 min at 72 °C, and 7 min sion 2.5 (10) to estimate ancestral states of characters illustrated extension at 72 °C. PCR amplicons were gel-excised using the in Fig. 2 and listed in Table S1, under a ML continuous-time QIAquick Gel Extraction Kit (QIAGEN) or purified directly Markov model (Mk1) on the ML tree shown in Fig. S1. Ances- using the QIAquick PCR Purification Kit (QIAGEN) following tral states were reported as proportional likelihoods at each the manufacturer’s instructions. Cycle-sequencing from both node for both character states. Missing data resulted in some strands was carried out on an ABI 3730 DNA Analyzer, Big Dye ancestral character states being reported as equivocal (Fig. S3). version 1.1 using ABI BigDye chemistry. Contiguous sequences were assembled and edited using Sequencher version 4.6 (Gen- Analysis of Correlated Evolution. We used BayesDiscrete in Bayes- eCodes Corp.), and sequence identity was checked using BLAST Traits (available at http://www.evolution.reading.ac.uk/BayesTraits. (http://www.ncbi.nih.gov/BLAST/). New sequences have been html) to test for correlated evolution between pairs of discrete deposited with GenBank under accessions FJ715295–FJ715334 binary character states (11). BayesTraits uses a reversible-jump inclusive (Table S3). Markov chain Monte Carlo (RJ MCMC) approach to search among possible models of character state evolution while sampling Phylogenetic Analysis. Alignments were performed in ClustalX (3) from a set of trees derived from a Bayesian phylogenetic analysis, using default settings and were improved by eye in MacClade thus taking phylogenetic uncertainty into account (11). The anal- (4). Regions that could not be aligned unambiguously were ex- ysis is run twice for each pair of character states, once allowing for cluded from the analysis. The full alignments for lsrDNA and dependent (or correlated) evolution (dep), and once restricting the ssrDNA gene partitions (with an indication of exclusion sets) are models to the null hypothesis of independent evolution (indep). available upon request. MODELTEST version 3.7maxX (5) was The rationale is that under independent evolution the transition used to select a model of evolution using the Akaike Information rate of character 1 from one state to the other should be in- Criterion. Phylogenetic trees were constructed using Bayesian in- dependent of the state of character 2 (11). The statistical inference ference (BI) with MrBayes version 3.1 (6) and using maximum compares the two analyses by the harmonic means (H)ofthe × − likelihood (ML) with PAUP* version 4.0b10 (7). For BI, likelihood resulting likelihoods using the test statistic 2 (logHdep log- > settings were set to number of substitution types (nst) = 6, Hindep). By convention, values for this test statistic 2 are taken rates = invgamma, ngammacat = 4 [equivalent to the general as positive evidence that the dependent model is favored, and time-reversible plus proportion of invariant sites plus gamma- values >5and>10 represent strong and very strong evidence, distributed rate variation across sites (i.e., GTR+I+G) model of respectively (11). nucleotide evolution]; parameters were estimated separately for We performed the analyses on a reduced taxon set containing each gene. Four chains (temp = 0.2) were run for 5 × 106 gen- only the genus Macrostomum, the main focus of our study. Spe- erations and sampled every 103 generations; 5 × 105 generations cifically, we tested if the character states for copulation behavior were discarded as burn-in. ML analyses were performed using (0, hypodermic; 1, reciprocal) correlate with those for sperm successive approximation: Model parameters were estimated bristles (0, absent; 1, present), or female antrum morphology (0, based on a starting tree determined by neighbor joining. A simple; 1, thickened). (Fig. 2, Fig. S3, and Table S1 give details on heuristic search was performed implementing the estimated character states.) Note that the character states for sperm bristles model parameters using nearest-neighbor–interchange branch and stylet morphology (0, needle-like; 1, not needle-like) are fully swapping. Model parameters were estimated on the best tree, congruent among the available Macrostomum species, so the re- and a heuristic search was performed using subtree-pruning- sults we present for sperm bristles also are valid for stylet mor- regrafting branch swapping. After model parameters were esti- phology. Moreover, the character states for the copulation mated, heuristic searches using tree-bisection-reconnection (TBR) behavior and the sucking behavior (0, never observed; 1, present) branch swapping were performed until the topology remained are nearly congruent, but the latter character has more un- unchanged. In addition to posterior probability values from BI certainty about its states (Table S1); here we focus only on the Schärer et al. www.pnas.org/cgi/content/short/1013892108 1of14 copulation behavior. Using this method we performed analysis A Gen. nov. 1, sp. nov. 1 (Macrostomidae) has been collected (sperm bristles vs. copulation behavior) and analysis B (female repeatedly by our group from a single location in Lignano, Italy antrum morphology vs. copulation behavior), each with a sepa- (45°41′28.5′′N, 13°07′54.0′′E). The location is on the shore of rate run for dependent and independent models. the Laguna di Marano below dense vegetation and consists of Based on initial ML runs, we used a RJ hyperprior with a gamma relatively clean sand with some humus content This genus distribution (rjhp gamma 0101)fortheRJMCMC analyses (11). probably belongs to a diffuse group of Macrostomidae that lack Next we optimized the rate deviation (ratedev) parameters to a sclerotized stylet, although currently it is unclear if these taxa achieve acceptance rates between 20–40% (11) and settled for form a monophyletic clade (24). These taxa might include My- 0.18 and 0.12, respectively, for the dependent and independent ozona Marcus 1949, Siccomacrostomum Schmidt and Sopott- runs of analysis A and 0.3 and 0.25, respectively, for the de- Ehlers 1976, Dunwichia Faubel, Bloome and Cannon 1994, pendent and independent runs of analysis B. We ran the analyses Psammomacrostomum Ax 1966, and Antromacrostomum Faubel with a sample of 500 best trees taken from our Bayesian phylo- 1974. Our species is clearly distinct from Myozona by the absence genetic analysis (performed as outlined above but with the re- of a muscular gizzard in the gut, from Siccomacrostomum by the duced taxon set) for 505 × 106 iterations with a burn-in of 5 × 106 absence of a common genital opening, from Dunwichia by the iterations and sampling period of 103 for the independent models presence of paired testes and ovaries, from Psammomacrostomum and for 1,020 × 106 iterations with a burn-in of 2 × 107 iterations by the presence of a vagina and female antrum, and from An- and sampling period of 4 × 103 for the dependent models (to tromacrostomum by the absence of an armed pharynx and the achieve reliable convergence stability). presence of a paired ovary. However, the copulatory organ in our species is similar to that of Psammomacrostomum equicaudatum, Notes on Species Identification, Sampling Locations, and and the close proximity of the male and female genital openings is Taxonomic Status of the Studied Specimens similar to Antromacrostomum armatum. Thus, these three genera We have deposited extensive digital reference material for all may be closely related.