Adult Alboglossiphonia lata, Alboglossiphonia sp., and Barbronia sp. specimens were collected by examining submerged plants, leaves, and plastic bags in selected localities of Bangjook reservoir in Cheongju, Chungcheongbuk-do (South Korea). Adult Glossiphonia sp. was collected in selected localities of Dal stream in Goesan-gun, Chungcheongbuk-do (South Korea). Adult Hemiclepsis sp. was collected in selected localities of Miho stream in Cheongju, Chungcheongbuk-do (South Korea). Helobdella austinensis was bred in the laboratory. All adult specimens except Glossiphonia sp. and Hemiclepsis sp., which cannot be incubated in artificial conditions, were incubated in a bowl containing artificial pond water. Glossiphonia sp. and Hemiclepsis sp. were fixed with 100% EtOH until used for the histological analysis. The specimens were cared for once daily by changing solution and the bowl was scrubbed manually to get rid of any residual waste. They were stored in a BOD incubator at 22 ℃.
CO1 gene cloning and sequencing
Total RNA was isolated from Alboglossiphonia sp. embryos using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). mRNA was purified from Total RNA with Oligo (dT) primer (Promega, Madison, WI, USA), and reverse transcribed into cDNA with a SuperScript II First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA USA). Genomic DNA was extracted using QIAamp DNA Mini Kit (QIAGEN, Hilden, NW, Germany). We amplified the A. lata CO1 gene sequences  and other leech-specific CO1 and 18S rRNA genes using universal primers . We used the TaKaRa Ex Taq® kit (Takara Bio Inc., Kusatsu, Japan) according to the manufacturer's instructions (predenaturation - 94℃, 5min; denaturation - 94℃, 30sec; annealing – variable, 30sec; extension - 72℃, 1min for COI or 1min 30sec for 18s rRNA sequence fragments; post extension - 72℃, 5min).
Three partial nucleotide sequences of the mitochondrial cytochrome c oxidase subunit 1 (CO1) (Alboglossiphonia sp., A. lata, and Barbronia sp., about 700 bp), and four partial sequences of 18S ribosomal RNA (18S rRNA, about 1.8 kb) from the same three species plus Helobdella austinensis were obtained in this study by PCR amplification. Additional sequences of both genes were obtained from the GenBank, and two alignments of 62 COI and 62 18S genes from the same group of species (see Additional file1: Table S1 for GenBank accession number) were prepared using ClustalW implemented in MEGA7 software (ver. 7.0.26) , and then concatenated. Phylogenetic tree hypotheses were prepared from the concatenated matrix using Maximum Likelihood (ML) and Bayesian Inference (BI). The best-fit model was searched based on the corrected Akaike Information Criterion (AICc) using IQ-TREE  web-server (http://www.iqtree.org). The ML and BI analyses were conducted using RAxML-NG software (v 0.9.0)  and MrBayes software (ver. 3.2.7a)  under the General Time Reversible model (GTR) with a proportion of invariable sites (I) and a gamma-shaped distribution rates (G4). The ML tree reconstruction was initially attempted by generating 3,000 bootstrap replicates with “autoMRE” command. The bootstrapping support values for branches were estimated under the transfer bootstrap expectation (TBE) . Markov Chain Monte Carlo (MCMC) for the BI tree was run with 5,000,000 generations and the BI tree was constructed by discarding the first 25% generations. The trees were visualized with FigTree software (ver. 1.4.4).
Prey selection test and tracking analysis
In order to compare feeding behaviors of leeches, we conducted a survey in the laboratory environment using various food types: Limnodrilus hoffmeisteri (Clitellata, Annelida), swallowable and worm shape; Biomphalaria sp. and Physella sp. (Gastropoda, Mollusca), unswallowable and carrying a shell; and Chironomus sp. (Insecta, Arthropoda), unswallowable and exhibiting a worm shape. First, several individuals of each leech species were placed in the 55 mm petri-dish, and ingestion patterns were observed under mixed prey species (single leech species vs. multiple prey species) and each prey species (single leech species vs. single prey species) to confirm exact preferences and ingestion behavior. After observation, one or two prey organisms were provided to each leech. Each experimental dish was video-recorded using a DCR-SR200 camcorder (SONY, Minato, TYO, Japan) over 8 hr, or until the leeches completed feeding under room temperature. Ingestion behavior tests were performed on three biological replicates in the same condition as described above. Among the recorded videos, location analysis on ingestion behavior was conducted using one representative video for each species. To analyze the behavior of both leeches and the prey, the location of all individuals present in the petri dish was tracked every 3 min using EthoVision software (Noldus Information Technology, Wageningen, GE, Netherlands). When the predators were supplied with two species of prey, only the behavior of prey that was ingested was tracked. However, when Barbronia sp. was provided with L. hoffmeisteri or Chironomus sp., the individual location was tracked every 30 s due to their relatively rapid ingestion. Distances between leeches or preys and a reference point established on the 12 o’clock edge of the petri-dish were recorded.
To visualize differentiation of proboscis muscle structure, adult leeches were treated with relaxation buffer (4.8 mM NaCl2, 1.2 mM KCl, 10 mM MgCl2, 8% EtOH) and fixed in 4% PFA (Electron Microscopy Sciences, Hatfield, PA, USA) in 1X phosphate buffered saline (PBS) overnight at 4°C. For H&E staining, leeches were dehydrated in EtOH series and cleared in Xylene (Central Drug House, New Delhi, DL, India) for 2 hr. The leeches were embedded in paraffin (Leica, Wetzlar, HE, Germany) and stored at –20°C. Paraffinized samples (10 µm thickness) were cut with a RM2235 microtome (Leica, Wetzlar, HE, Germany) and stained with Mayer’s Hematoxylin (Cancer Diagnostics, Durham, NC, USA) and Eosin (Cancer Diagnostics, Durham, NC, USA). Samples were mounted on glass slides with an Organo Mount (ImmunoBioScience, Mukilteo, WA, USA) and dried overnight at room temperature. Sections were imaged with a LEICA DM6 B compound light microscope (Leica, Wetzlar, HE, Germany) and a LEICA DFC450 C camera (Leica, Wetzlar, HE, Germany). The obtained images were edited using Las X software (Leica, Wetzlar, HE, Germany) and Adobe Photoshop CS5 (Adobe, San Jose, CA, USA). The edited images were prepared as figure plates using Adobe Illustrator CS6 (Adobe, San Jose, CA, USA). To obtain cryo-sections, leeches were embedded in O.C.T. compound (VWR, Radnor, PA, USA) and rapidly frozen in liquified nitrogen. Cryo-sectioned samples (15 µm in thickness) were cut with a CM1520 cryostat (Leica, Wetzlar, HE, Germany) and stored at –70°C until use.
Scanning electron microscopy of proboscis feeding organs
For scanning electron microscopy, leech specimens were treated with 16% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) or relaxation solution (4.8 mM NaCl2, 1.2 mM KCl, 10 mM MgCl2, 8% EtOH) while feeding or relaxing. After treatment, the head region containing the proboscis was cut and fixed in 4% PFA at room temperature for overnight. The tissues were washed three times with PBT (1X PBS + 0.1% Tween-20) for 20 min at room temperature, and then fixed in 1% osmium tetroxide (Ted Pella Inc., Redding, CA, USA) in 1M PBS for 1 hr. Osmium tetroxide was removed by washing three times with PBT. Thereafter, the tissues were gradually dehydrated with ethanol (30%, 50%, 60%, 70%, 80%, 90%, 95%, 100% in 1X PBS) for 20 min per step. Dehydrated tissues were treated with stepwise concentrated isopentyl acetate (Alfa Aesar, Ward Hill, MA, USA) (isopentyl acetate: EtOH = 1:3, 1:1, and 3:1) for 15 min per step, and then transferred to 100% isopentyl acetate. After the solution was removed, the samples were dried for 3 days in the hood. Dried samples were coated with gold particle and examined with an UltraPlus field emission scanning electron microscope (Carl Zeiss, Oberkochen, BW, Germany).
Fluorescent labeling and immunohistochemistry
Whole-mount immunostaining was performed according to previously published protocols , with the following details: The cross-sections were dried and washed in PBT (0.1% Tween-20 with 1X PBS) five times. The nerve and muscle fibers were visualized after double immunostaining as follows. After washing with PBT, the sections were incubated in diluted blocking solution (1:9 = 10X Roche Western Blocking Reagent : PBT) for 2 h. Samples were incubated with primary antibodies (anti-acetylated-α-Tubulin produced in mouse, Sigma Aldrich, T-7451; or anti-cardiac TroponinT produced in rabbit, Abcam, ab115134) in diluted blocking Solution (1:500) at 4 ℃ for 48 h. After five consecutive washes with PBT, the sections were incubated with a secondary antibody (goat anti-mouse IgG H&L Alexa Fluor 488, Abcam, ab150113; goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody Alexa Fluor 568, Invitrogen, A11011) in diluted blocking Solution (1:1000) at 4 ℃ for 24 h. After checking the labeled signal, the samples were washed five times with PBT, and then stained with Texas Red™-X Phalloidin (ThermoFisher, T7471) for 1 h to visualize F-actin. After checking the labeled signal, the samples were washed five times with PBT and labeled with DAPI in PBT (1:100) at room temperature in the dark overnight. After washing with PBT five times, the samples were mounted with Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Fluorescence-stained embryos and slide samples were imaged using a LEICA DM6 B with a LEICA DFC450 C camera (Leica, Wetzlar, HE, Germany). The obtained images were edited using Las X software (Leica, Wetzlar, HE, Germany). To confirm the detailed muscle structure and innervation in the proboscis, slides co-labeled with F-actin and acetylated tubulin were imaged with a LSM 710 confocal microscope (Carl Zeiss, Oberkochen, BW, Germany). The obtained images were edited using ZEN software (L Carl Zeiss, Oberkochen, BW, Germany). The edited images were prepared as figure plates using Adobe Illustrator CS6 (Adobe, San Jose, CA, USA).
ST-MHC gene identification, probe synthesis and in situ hybridization
Total RNA was isolated from H. austinensis mixed-stage embryos and Hirudo nipponia head tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). We selected mRNA from total RNA using Oligo (dT) primer (Promega, Madison, WI, USA) and synthesized cDNA (SuperScript II First Synthesis System for RT-PCR, Invitrogen, Carlsbad, CA, USA). To isolate the H. austinesis striated myosin heavy chain (ST-MHC) gene, a previously published sequence  was used and screened using a BLAST implemented in the whole draft-genome reference (http://genome.jgi.doe.gov/Helro1/Helro1.home.html). Two candidate genes (protein id 64397 and 129847) were screened, and the foregut specific st-mhc gene was isolated by confirming the foregut specific expression pattern (protein id: 129847) (Additional file 1: Fig S3B). In the H. nipponia transcriptome data, only a single striated myosin heavy chain transcript was found, which showed a high degree of similarity to the H. austinensis foregut specific st-mhc gene (nucleotide similarity: 81%, translated sequence similarity: 93%) (For nucleotide similarity, see Additional file 1: Fig S3C). The st-mhc specific primers were designed to amplify the consensus region of the two sequences producing similar length (product sizes about 850 nucleotides - protein id 129847, Hau st-mhc forward: 5’-GCCACCAAAGGTGAAGAG-3’; Hau st-mhc reverse: 5’-GTCCTCAACGAGCTGCAT-3’). H. nippoinia st-mhc transcript (Hni st-mhc forward: 5’- GCCACCAAGGGCGAAGAA-3’; Hni st-mhc reverse: 5’- TCCTCGACCAATTGCATTTCC-3’). These amplified fragments were cloned into pGEM T vector (Promega, Madison, WI, USA). RNAprobes labeled with digoxigenin were made using the MEGAscript kit (Ambion, Austin, TX, USA) and DIG RNA Labeling Mix (Roche, Basel, Switzerland), according to the manufa cturer's instructions. The synthesized RNA probes were applied to each sample at a final concentration of 2 ng/μl, and the probe labeled samples were incubated with an Anti-Digoxigenin-POD Fab fragments produced in sheep (Roche, Basel, Switzerland) in diluted blocking solution (1:1000). The detail procedure of in situ hybridization was followed using previously published methods [54, 65, 66]. After cryosection, stored samples were dried to remove residual moisture. Dried samples were treated with 0.2N HCl buffer to inhibit endogenous enzymes and rinsed three times with PBT. After this process, the following experiments were carried out using the same protocol as described above.