Redescription of the Siamese shield leech Placobdelloides siamensis with new host species and geographic range

The Siamese shield leech Placobdelloides siamensis (Oka, 1917) Sawyer, 1986 (Euhirudinea: Glossiphoniidae) was collected from five new host species, Southeastern Asian Box Turtle (Cuora amboinensis), Yellow-headed Temple Turtle (Heosemys annandalii), Malayan Snail-eating Turtle (Malayemys macrocephala), Mekong Snail-eating Turtle (M. subtrijuga), and Khorat Snail-eating Turtle (M. khoratensis) and was found for the first time in Udon Thani, Thailand. Examination of live leeches provided, for the first time, data on coloration and the combination of parental care behavior, both carrying cocoons and attaching cocoons to the substrate. This species was separated from its congeners based on the following characters: one pair of eyes; spines at proboscis subterminal; mouth terminal on oral sucker; absent plaque in neck region; gonopores located in furrow and separated by two annuli; distinctly triannulated mid-body segments; crop with seven pairs and branched caeca; caudal sucker slightly over half of maximum body width; and strongly dorsal papillae. Phylogenetic relationships based on the COI and ND1 genes were clarified and demonstrated that the species is distinct from others. The original description was amended and the taxonomic history is discussed.

In Thailand, turtles are commonly used in the practice of religious merit release and are believed to grant a long life to the releaser. Frequently found species of turtles include Southeast Asian Box Turtle (Cuora amboinensis Daudin, 1802), Yellow-headed Temple Turtle (Heosemys annandalii Boulenger, 1903) [3,7,23]. There have been almost no reports on their infection with leeches before; however, in this study, these turtles from various areas were found to be heavily infected with freshwater leeches, subsequently identified as Placobdelloides siamensis.
The Siamese shield leech P. siamensis was originally described in the early 20th century as Hemiclepsis siamensis Oka, 1917 from the black marsh turtle Bellie crassicollis Gray, 1831 (currently, S. crassicollis), which originated from Lampam, Patalung, Thailand, before having been reclassified to the current name by Sawyer in 1986 [1,5,20]. Even though Siddall et al. [21] have previously sequenced and reported this species from Wat Bovorn, Bangkok, Thailand, they did not describe the host species, nor did they update the data. Therefore, in this study, we examined information on P. siamensis to expand the original description by updating the known morphology and host range in Thailand, and including the ban on the release of turtles to prevent the spread of P. siamensis.

Molecular analysis
Leeches were cut into two pieces equally; the anterior part was kept in an absolute ethanol. Posterior part was used for DNA isolation with the DP304-02 TIANamp Genomic DNS, following the protocol given for the purification of total DNA from animal tissue (spin column). For the proteinase K treatment step, tissue samples were lysed for several hours at 56°C. DNA was eluted from the spin column with 150 lL of buffer.

Statistical analysis
PCR products were purified and sequenced using either the HCO2198 and LCO1490 primers for the CO-I products or the HND1932 and LND300 for the ND-I products by Macrogen Korea. The sequences obtained were submitted to GenBank (Table 1). Comparative sequences were retrieved from GenBank. The DNA sequences were aligned using ClustalW version 1.83 software and analyzed using MEGA6 version 6 software for Maximum likelihood analyses, and MrBayes version 3.1.2 software for Bayesian analysis.
Maximum likelihood analyses consisted of 2000 replicates of tree search with 25 initial GAMMA rate categories and final optimization using four GAMMA shape categories; bootstrap values were calculated using 2000 pseudoreplicates of the rapid bootstrap algorithm. And Bayesian analysis was run for 2 million generations with tree sampled every 100 generations, with the general time reversible (GTR) model and GAMMA distribution of nucleotide rates for all partitions. Burn-in was set to 10%.

Description
Specimens were determined to belong to Placobdelloides 1986 and were characterized as triannulate in the mid-body, with one pair of eyes on somite III, a terminal mouth pore, a crop with seven pairs of gastric ceca, a gonopore between two annuli, a caudal sucker slightly over the half body width, and cocoons and eggs attached directly to both the ventral surface of an adult and to the substrate [1,8,20].

External morphology
Body of mature P. siamensis an elongated oval, 25 mm in length, maximum head and body width 2.35 and 5.57 mm, respectively (Fig. 1). Entire dorsal surface rough, with numerous well-developed 5-9 longitudinal rod papillae present. No regular arrangement pattern, except a row of papillae on each side of the median line. Ventral surface entirely smooth.
Oral sucker small (average 1.86 mm diameter), with mouth opening at terminal portion and numerous pits inside ( Fig. 2A). One pair of eyes, located on dorsal surface of somite III (Fig. 2B). Eyes touch. Male genital pore situated in furrow between annuli 22 (3rd ring of somite X) and 23 (1st ring of somite XI) (Fig. 3). Female pore lies in furrow of somite XI between annuli 24 (1st ring) and 25 (2nd ring). Two annuli separate the gonopores, and anus separates last annulus and caudal sucker. Caudal sucker over half of body width (average 3.00 mm in diameter), no pits.

Internal anatomy
Blunt elongated cylindrical proboscis from terminal lip of oral sucker to posterior proboscis sheath, followed by elongated esophagus, to which discrete compact bunch of salivary glands and diverticulate esophagus glands open. Seven pairs of diverticulated crop ceca, with last pair extending posteriorly and diverticulated into four sections. Four pairs of simple saccular intestinal, with last pair extending posteriorly to simple rectum and anus. Anus opens on dorsal surface behind the last annulus (Fig. 4).
Male genital pore opens to glasses shaped sac, posteriorly to the diverticulate esophagus, and diverts to vas deferens on each side. Vas deferens posterior, opens to six pairs of curly rounded testes, each located close to the distal edge of each crop branch. Female pore situated posteriorly to male genital pore, opens to a sac which continues into elongated ovary on each side. Typically, ovaries coiled anteriorly the first crop caeca; however, after mating and egg producing, ovaries are elongated posteriorly from numerous eggs inside them.

Molecular analysis
The Bayesian trees of the COI-ND1 genes of the Glossiphoniid leeches had high probability support values for the monophyly of AN000016CE, AN000017CE, AN000018CE, AN000021CE, AN000022CE, AN000023CE, P. siamensis, and P. Jaegerskioeldi, but also revealed non-monophyly of P. multistriatus (Fig. 6). The maximum-likelihood trees also yielded monophyletic groups, similar to the Bayesian trees, but the data were different for P. jaegerskioeldi and P. multistriatus. The barcoding results also confirmed that AN000016CE, AN000017CE, AN000018CE, AN000021CE, AN000022CE, and AN000023CE were P. siamensis.

Discussion
This is the first record of P. siamensis from C. amboinensis, H. annandalii, M. macrocephala, M. subtrijuga, and M. khoratensis. This leech has currently been recorded from six species of turtles from the same family (Geoemydidae). In addition, this is the first record of P. siamensis from Udon Thani (northeastern province) in Thailand. The leech was previously reported only in Patalung (southern province) and Bangkok (central province) in Thailand, as well as in Nanking, China [20,21]. Even though this leech has been shown to feed on numerous Geoemydidae turtles, as mentioned above, there have been no records that it is able to feed on other families, as found in this study. Moreover, it tends to spread throughout Thailand following its host distributions.
The morphological characters that separated this P. siamensis from it congeners are one pair of eyes; spines at the proboscis subterminal; mouth terminal on oral sucker; absent plaque in neck region; gonopores located in furrow and separated by two annuli; distinctly triannulated mid-body segments; crop with seven pairs and branched ceca; caudal sucker slightly over half maximum body width; and strongly dorsal papillae. The specimens collected in this study almost   matched the description of P. siamensis (in alcohol) by Annandale [1], except the male and female gonopores. The male gonopore had been described as situated between annuli 25 and 26 (somite XI and XII), while it was situated between annuli 22 and 23 (somite X and XI) in this study.
The female gonopore had been described as situated between annuli 27 and 28 (somite XII), while it was situated between annuli 24 and 25 (somite XI) in this study. Since Annandale [1] used preserved specimens, no information on coloration in life was provided. The report by Siddall et al. [21] on the sequencing of P. siamensis from Wat Bovorn did not describe morphological details, nor the host. Collection of a large number of live leeches in this study allowed us to describe the external coloration and morphology.
All specimens examined were mature, with developed testisacs and ovisacs present. Some specimens carried cocoons  (Table 2): the upper diagram is the Bayesian analysis; lower is the maximum likelihood analysis.  or young on the posterior ventral surface, as in the original description. In this study, some cocoons were attached to the substrate. The number of eggs was documented to be between 173 and 412 per individual in 14 specimens.
In the original description, Annandale [1] hesitated to classify this species into Hemiclepsis or Placobdella because of the oval shape, one pair of eyes, a pattern of numerous irregular papillae, gonopores separated by two annuli, and cocoons carried on the ventral side. Finally, it was placed in Hemiclepsis based on the distinctly posterior position of the mouth, although Hemiclepsis is characterized by 9-11 pairs of crop ceca, 2-3 pairs of eyes, gonopores separated by two annuli, and the attachment of cocoons to the substrate [20].
Sawyer [20] subsequently moved H. siamensis to P. siamensis because of the following similarities: one pair of eyes, an esophageal organ, seven pairs of crop ceca, a terminal mouth pore, gonopores separated by two annuli, and a combination of the parental care behavior, either carrying cocoons on the ventral surface or attached to the substrate.
Although Annandale [1] and Sawyer [20] clearly described the morphology of P. siamensis, this study provides a more complete overview of the morphological structure, coloration pattern, and parental care behaviors of P. siamensis, thus leading to a better understanding of its actual phylogenetic position within glossiphoniid leeches.