Corresponding author: Takafumi Nakano (
Academic editor: Fredric Govedich
A new quadrannulate species of
Nakano T (2014) A new quadrannulate species of
The genus
Several small
Leeches were collected from seven localities in east-central Honshu, Japan (Fig.
Map showing the collection localities of the specimens examined in this study. Open circle (4) indicates the
Collection localities in this study with the information on locality names.
Locality number | Locality name |
---|---|
1 | Akiruno, Tokyo Metropolis, Japan |
2 | Namesawakeikoku Valley, Izu Shizuoka Prefecture, Japan |
3 | Shibunoyu, Kitayama, Chino, Nagano Prefecture, Japan |
4 | Mt. Mitsugaisan, Ina, Nagano Prefecture, Japan |
5 | Shirabisotoge Pass, Ida, Nagano Prefecture, Japan |
6 | Ikuta, Matsukawa, Nagano Prefecture, Japan |
7 | Shiojidaira Nature Park, Iizuna, Nagano Prefecture, Japan |
The specimens were relaxed by the gradual addition of absolute ethanol to fresh water. For DNA extraction, botryoidal tissue was taken from the posterior part of the body around the caudal sucker of every specimen, and then preserved in absolute ethanol. The rest of the body was fixed in 10% formalin and then preserved in 70% ethanol. Four measurements were taken: body length (BL) from the anterior margin of the oral sucker to the posterior margin of the caudal sucker, maximum body width (BW), caudal sucker length (CL) from the anterior to the posterior margin of the sucker, and caudal sucker width (CW) from the right margin to the left margin of the sucker. Examination, dissection, and drawing of the specimens were accomplished using a stereoscopic microscope with a drawing tube (Leica M125). Specimens used in this study have been deposited in the Zoological Collection of Kyoto University (KUZ).
The numbering convention is based on
The extraction of genomic DNA from botryoidal tissues preserved in absolute ethanol followed
Samples used for the phylogenetic analyses. The information on the vouchers is accompanied by the collection locality numbers for
Species | Voucher | 18S | Histone H3 | COI | tRNACys–16S | tRNALeu–ND1 |
---|---|---|---|---|---|---|
KUZ Z694 Holotype (4) |
|
|
|
|
|
|
KUZ Z684 (1) |
|
|
|
|||
KUZ Z687 (2) |
|
|
|
|||
KUZ Z689 (5) |
|
|
|
|||
KUZ Z696 (7) |
|
|
|
|||
KUZ Z697 (6) |
|
|
|
|||
KUZ Z699 (3) |
|
|
|
|||
KUZ Z120 Holotype | ||||||
KUZ Z29 Holotype | ||||||
KUZ Z110 Topotype | ||||||
KUZ Z167 Topotype | ||||||
KUZ Z208 Holotype | ||||||
KUZ Z156 Holotype | ||||||
KUZ Z224 Holotype | ||||||
KUZ Z181 Topotype | ||||||
KUZ Z128 Holotype | ||||||
KUZ Z134 Holotype | ||||||
KUZ Z45 Topotype | ||||||
KUZ Z178 | ||||||
UNIMAS/A3/BH01/10 | ||||||
KUZ Z179 | ||||||
KUZ Z180 |
|
|
|
|
Sources: a
Sixty-six previously published sequences (
The phylogenetic position of the new species within the genus
Phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI) models. ML phylogenies were calculated using TREEFINDER v. October 2008 (
The phylogenetic relationships of the specimens of the new species were reconstructed based on sequences of mitochondrial regions. The alignment of the sequences as well as the reconstruction of the ML and BI phylogenies was accomplished followed the methods described above. The length of the aligned COI was 1266 bp, that of tRNACys–16S was 1056 bp, and that of ND1 was 579 bp. Thus, the concatenated sequences yielded 2,901 bp positions. The best-fit models for each partition selected for the ML phylogenies were as follows: for the first, second, and third positions of COI, respectively, TN93+G, TVM+H, and TN93+G; for tRNACys–16S, GTR+G; and for the first, second, and third positions of ND1, respectively, TN93+G, HKY85+H, and HYK85+G. The best-fit models identified for each partition for the BI analyses were as follows: the first, second, and third positions of COI, respectively, GTR+G, F81+H, and HKY85+G; for tRNACys–16S, GTR+G; and for the first, second, and third positions of ND1, respectively, GTR+G, F81+H, and HKY85+G. For BI and BPPs, two independent runs of four Markov chains were conducted for 6 million generations, and the tree was sampled every 100 generations. The first 15,001 trees were eliminated based on the results of the parameter estimates and convergence.
Nodes with BS values higher than 70% were considered sufficiently resolved (
Pairwise comparisons of the Kimura-2-parameter (K2P) distance (
Body length of mature individual less than 4 cm. Somite IV uniannulate, somites VIII–XXV quadrannulate. Clitellum in XI b5 to XIII a2. Pharynx reaching to XIV. Gastropore conspicuous in middle of XIII a1. Gastroporal duct bulbous, winding at junction with gastropore. Male gonopore in middle of XI b6, female gonopore inconspicuous in middle of XIII a1, behind gastropore, gonopores separated by 1/2 + 4 + 1/2 annuli. Paired epididymides in XV/XVI–XVI b5/b6 to XVII b5/b6–XVIII/XIX, occupying 7–10 annuli (i.e. one and a half to two and a half somites). Atrial cornua developed, ovate.
In total, 11 specimens examined. KUZ Z684–Z686 (three specimens), collected from under rocks in Akiruno (locality number 1), by TN: KUZ Z684, from along a mountain trail at Mt. Kariyoseyama (
The specific name is a noun in the genitive case formed directly from the name of Mr Masaaki Kuroiwa, who generously accompanied the field survey in Nagano Prefecture.
Body firm and muscular, elongate, with constant width in caudal direction, dorsoventrally compressed, BL 34.0 mm, BW 3.42 mm (Fig.
Somite I completely merged with prostomium (Fig.
Anterior ganglionic mass in VI a2 and a3. Ganglia VII–X, of each somite, in a2 (Fig.
Eyes in three pairs, first pair dorsally on anterior margin of III, second and third pairs dorsolaterally on posterior margin of V (a1 + a2) (Fig.
Pharynx agnathous, euthylaematous, reaching to XIV a1/a2 (Fig.
Male gonopore in middle of XI b6 (Fig.
BL 22.4 (KUZ Z686) –35.2 (KUZ Z684) mm, BW 2.3 (KUZ Z691) –3.5 (KUZ Z684) mm, CL 1.1 (KUZ Z686)–1.7 (KUZ Z693) mm, CW 1.1 (KUZ Z686)–2.1 (KUZ Z689) mm. Somites III, IV uniannulate, each with slight dorsal furrow (KUZ Z695). Somite XXVI variable; often dorsally quadrannulate, ventrally triannulate, rarely with slight ventral furrow in a3; KUZ Z699 with quadrannulate; KUZ Z698, Z691 with triannulate with slight dorsal furrow in a3; KUZ Z689 with triannulate. Somite XXVII biannulate, or uniannulate with slight dorsal furrow. Eyes in three pairs; KUZ Z699 with one eye dorsoleft on posterior margin of III. Pharynx reaching to XIII/XIV–XIV a2/b5. Crop reaching to XIX b5/b6–XX a1. Gastropore occasionally slightly posterior to middle of XIII a1. Gastroporal duct joining with crop in XIV a1/a2–XIV b6; KUZ Z687 with thick, tubular duct. Intestine reaching to XXIII/XXIV–XXV a2. Male gonopore rarely slightly anterior to middle of XI b6, or slightly posterior to middle of XI b6. Female gonopore occasionally slightly posterior to middle of XIII a1. Testisacs in XVII b6–XIX a1 to XXIV b5–XXV a2. Epididymides in XV/XVI–XVI b5/b6 to XVII b5/b6–XVIII/XIX; occupying 7–10 annuli. Atrial cornua generally ovate; KUZ Z696 ellipsoid; KUZ Z687 fusiform. Pre-atrial loop absent, or reaching to middle of XI b5 (KUZ Z693, Z697). Ovisacs often in XIII a2–b6; KUZ Z687, Z699 in XIII a2, b5; KUZ Z696 right one in XIII a2–XIV a1/a2, left one in XIII a2–XIV a1. Right or left oviduct crossing ventrally beneath nerve cord; KUZ Z684, Z693 both oviducts converging into common oviduct in XIII a2.
In life, dorsal surface ochre (Fig.
This species was primarily collected from localities in Nagano Prefecture: the east-central part (locality number 3), and the southeastern part along the Inadani Basin (locality numbers 4–7). This species was also found in the western mountainous part of the Metropolitan Tokyo area (locality number 1), as well as in the Amagi Mountain Range in the central part of the Izu Peninsula, Shizuoka Prefecture (locality number 2). The locality data for this species suggested that
This species was generally found curled up under rocks or fallen leaves in moist mountainous habitats (Fig.
Mature leeches with an obvious clitellum were collected on 20 July (KUZ Z690, Z691, Z693, Z694) and 10 August (KUZ Z697) at two sites in Nagano Prefecture (locality numbers 4 and 7, elevation ca. 875 m and 1098 m, respectively). These findings indicate that the reproductive season of this species may begin in mid-to-late July.
Although the leech specimens examined in this study were small (up to 35 mm), several individuals, including the holotype, were determined to be mature due to the possession of an obvious clitellum and developed testisacs. Specimen KUZ Z687 possessed a tubular gastroporal duct and fusiform atrial cornua. Immature leeches may have these characteristics, because the sperm ducts and testisacs of specimen KUZ Z687 are undeveloped and barely detectable.
The new species unambiguously belongs to the genus
According to previous taxonomic studies (
Comparison of morphological characters between
|
|
||||||
|
less than 4 cm | up to ca 10 cm | up to ca 10 cm | up to ca 10 cm | less than 4 cm | up to ca 10 cm | up to ca 10 cm |
|
uniannulate | uniannulate | biannulate | uniannulate | uniannulate | uniannulate | uni- or biannulate |
|
1/2 + 4 + 1/2 | 2/3 + 4 + 1/3 | 6 | 1/2 + 4 + 1/2 | 1/2 + 4 + 1/2 | 1/2 + 5 | 1/2 + 4 +1/2 |
|
quadrannulate | quadrannulate | quadrannulate | quadrannulate | triannulate | quadrannulate | quadrannulate |
|
bulbous | tubular, but bulbous at junction with gastropore | simple tubular | simple tubular | bulbous | bulbous | bulbous |
|
XVI to XVIII | XVI to XX | XVI to XVII | absent | XV to XX | XVII to XIX | XVI to XVIII |
|
ovate | ovate | undeveloped | undeveloped | ovate | ovate | ovate |
The quadrannulate
The ML tree (ln
The ML tree (ln
The ML tree (ln
The ML tree (ln
The COI K2P distance within
Kimura-2-parameter distances for the 1266 bp for the COI sequences of
Specimen (locality number) | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
---|---|---|---|---|---|---|---|
1: KUZ Z684 (1) | |||||||
2: KUZ Z687 (2) | 0.046 | ||||||
3: KUZ Z689 (5) | 0.065 | 0.061 | |||||
4: KUZ Z694 (4) | 0.065 | 0.058 | 0.033 | ||||
5: KUZ Z696 (7) | 0.063 | 0.059 | 0.035 | 0.005 | |||
6: KUZ Z697 (6) | 0.066 | 0.067 | 0.006 | 0.034 | 0.034 | ||
7: KUZ Z699 (3) | 0.063 | 0.059 | 0.027 | 0.023 | 0.023 | 0.027 |
The current molecular phylogenies showed that the specimens morphologically identified as the new species form a monophyletic group with strong support values. In addition, the K2P genetic distance of the COI sequences detected within the specimens was 0.5–6.7% (mean = 4.4%).
The author is grateful to Masaaki Kuroiwa for his generous assistance with my field survey in Nagano Prefecture. I am also grateful to Yume Imada (Kyoto University; KU) and Yoshiko Yamane (KU) for providing specimens of the new species, and to two anonymous reviewers and Dr Fredric R. Govedich (Southern Utah University) for their constructive comments on this manuscript. A part of this study was financially supported by Grants for Biodiversity and Evolutionary Research of Global COE (A06) and for Excellent Graduate Schools, both from MEXT, Japan, to Kyoto University, and JSPS Grant-in-Aid for JSPS Fellows and Young Scientists (B) (#26840127) to the author.