Research Article |
Corresponding author: Takafumi Nakano ( nakano@zoo.zool.kyoto-u.ac.jp ) Academic editor: Michael Ohl
© 2016 Takafumi Nakano.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Nakano T (2016) Four new species of the genus Orobdella from Shikoku and Awajishima island, Japan (Hirudinida, Arhynchobdellida, Orobdellidae). Zoosystematics and Evolution 92(1): 79-102. https://doi.org/10.3897/zse.91.7616
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Four new species of the genus Orobdella Oka, 1895 from the mountainous regions of Shikoku and Awajishima island, Japan are described. These new species consist of one quadrannulate, two sexannulate and one octannulate species. The quadrannulate Orobdella brachyepididymis sp. n. is a small species with a body length reaching only ca. 5 cm. The sexannulate Orobdella okanoi sp. n. was collected from Shikoku, and the other sexannulate species, Orobdella yamaneae sp. n., inhabits Awajishima island. The octannulate Orobdella nakahamai sp. n. is a large species with a body length greater than 20 cm and is only the second large octannulate species known within this genus. Phylogenetic analyses using nuclear 18S rRNA and histone H3, as well as mitochondrial cytochrome c oxidase subunit I, tRNACys, tRNAMet, 12S rRNA, tRNAVal, 16S rRNA, tRNALeu and NADH dehydrogenase subunit 1 markers, indicated that O. brachyepididymis is a sister species of the quadrannulate O. naraharaetmagarum Nakano, 2016, while the other three new species formed a clade closely related to O. masaakikuroiwai Nakano, 2014 and O. whitmani Oka, 1895. The ranges of the distant phylogenetic lineage groups of Orobdella overlap in Shikoku and adjacent islets.
Hirudinea , Orobdella , new species, gastroporous, molecular phylogeny, Japan
The genus Orobdella Oka, 1895 is a terrestrial macrophagous leech taxon that inhabits the Japanese Archipelago, Korean Peninsula and Taiwan (
Additionally, these 13 species can be also split into another three types based on the body lengths of mature leeches (
Additional Orobdella leeches were collected from Shikoku and Awajishima island, Japan. The specimens collected from Shikoku comprised three morphological units: a quadrannulate small-type, a sexannulate middle-type and an octannulate large-type. Meanwhile, Orobdella leeches on Awajishima island belonged to the sexannulate middle-type. Each of these four units is described here as a new species. In addition, the phylogenetic positions of these new species were estimated using nuclear 18S rRNA and histone H3, as well as mitochondrial cytochrome c oxidase subunit I, tRNACys, tRNAMet, 12S rRNA, tRNAVal, 16S rRNA, tRNALeu, and NADH dehydrogenase subunit 1 sequence data.
Leeches were collected from five localities in Shikoku, and one locality on Awajishima island, Japan (Fig.
Map showing the collection localities of the specimens examined in this study. The closed diamond (2) denotes the locality of Orobdella brachyepididymis sp. n., closed squares (4–6) show the localities of Orobdella nakahamai sp. n., closed triangles (3, 5) designate the localities of Orobdella okanoi sp. n., and the closed circle (1) specifies the locality of Orobdella yamaneae sp. n. Symbols in red indicate the type locality of each of the new species.
Almost all of the specimens were relaxed by the gradual addition of absolute ethanol to freshwater. For DNA extraction, botryoidal tissue was removed from the posterior part of the body around the caudal sucker of every specimen, and then preserved in absolute ethanol. The remainder of the body was fixed in 10% formalin and 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 to the left margin of the sucker. Examination, dissection, and drawing of the specimens were conducted using a stereoscopic microscope with a drawing tube (Leica M125). Specimens used in this study have been deposited in the Zoological Collection of
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 the new species of Orobdella described in this study (see Fig.
Species | Voucher (locality number) | INSDC # | GenSeq Nomenclature | ||||
---|---|---|---|---|---|---|---|
18S | Histone H3 | COI | tRNACys–16S | tRNALeu–ND1 | |||
Orobdella brachyepididymis sp. n. |
|
LC106319* | LC106321* | LC106320* | LC106318* | LC106322* | genseq-1 18S, H3, COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1 |
Orobdella brachyepididymis sp. n. |
|
LC106324* | LC106323* | LC106325* | genseq-2 COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1 | ||
Orobdella nakahamai sp. n. |
|
LC106327* | LC106326* | LC106328* | genseq-2 COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1 | ||
Orobdella nakahamai sp. n. |
|
LC106330* | LC106332* | LC106331* | LC106329* | LC106333* | genseq-1 18S, H3, COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1 |
Orobdella nakahamai sp. n. |
|
LC106335* | LC106334* | LC106336* | genseq-3 COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1 | ||
Orobdella okanoi sp. n. |
|
LC106338* | LC106337* | LC106339* | genseq-3 COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1 | ||
Orobdella okanoi sp. n. |
|
LC106341* | LC106343* | LC106342* | LC106340* | LC106344* | genseq-1 18S, H3, COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1 |
Orobdella yamaneae sp. n. |
|
LC106346* | LC106345* | LC106347* | genseq-2 COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1 | ||
Orobdella yamaneae sp. n. |
|
LC106349* | LC106351* | LC106350* | LC106348* | LC106352* | genseq-1 18S, H3, COI, tRNACys, tRNAMet, 12S, tRNAVal, 16S, tRNALeu, ND1 |
Orobdella dolichopharynx Nakano, 2011b |
|
AB663665 | AB698876 | AB679680 | AB679681 | AB828558 | |
Orobdella esulacata Nakano, 2010 |
|
AB663655 | AB698873 | AB679664 | AB679665 | AB828555 | |
Orobdella ijimai Oka, 1895 |
|
AB663659 | AB698877 | AB679672 | AB679673 | AB828559 | |
Orobdella kawakatsuorum Richardson, 1975 |
|
AB663661 | AB698878 | AB679704 | AB679705 | AB828561 | |
Orobdella ketagalan Nakano & Lai, 2012 |
|
AB704785 | AB704786 | AB704787 | AB828582 | AB828563 | |
Orobdella koikei Nakano, 2012b |
|
AB698883 | AB698882 | AB679688 | AB679689 | AB828560 | |
Orobdella masaakikuroiwai Nakano, 2014 |
|
AB938003 | AB938013 | AB938006 | AB937997 | AB938016 | |
Orobdella mononoke Nakano, 2012a |
|
AB698868 | AB698869 | AB698866 | AB698867 | AB828564 | |
Orobdella naraharaetmagarum Nakano, 2016 |
|
LC087143 | LC087145 | LC087144 | LC087142 | LC087146 | |
Orobdella octonaria Oka, 1895 |
|
AB698870 | AB698871 | AB679708 | AB679709 | AB828562 | |
Orobdella shimadae Nakano, 2011b |
|
AB663663 | AB698875 | AB679676 | AB679677 | AB828557 | |
Orobdella tsushimensis Nakano, 2011a |
|
AB663653 | AB698872 | AB679662 | AB679663 | AB828554 | |
Orobdella whitmani Oka, 1895 |
|
AB663657 | AB698874 | AB679668 | AB679669 | AB828556 | |
Erpobdella japonica Pawłowski, 1962 |
|
AB663648 | AB698879 | AB679654 | AB679655 | AB828542 | |
Gastrostomobdella monticola Moore, 1929 | UNIMAS/A3/BH01/10 | AB663649 | AB698880 | AB679656 | AB679657 | AB828543 | |
Mimobdella japonica Blanchard, 1897 |
|
AB663650 | AB698881 | AB679658 | AB679659 | AB828544 | |
Odontobdella blanchardi (Oka, 1910a) |
|
AB663651 | AB938012 | AB938004 | AB937995 | AB938014 |
Eighty-five published sequences were obtained from the INSDC for use in molecular phylogenetic analyses (Table
The phylogenetic relationships of the newly identified Orobdella species within the genus was estimated based on 18S, H3, COI, tRNACys–16S and tRNALeu–ND1 sequences. The alignments of H3 and COI were trivial, as no indels were observed. 18S, tRNACys–16S, and tRNALeu–ND1 were aligned using MAFFT v. 7.266 L-INS-I (
Phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI). ML phylogenies were constructed using RAxML v. 8.1.5 (
BI and Bayesian posterior probabilities (BPPs) were estimated using MrBayes v. 3.2.5 (
Nodes with bootstrap support (BS) values higher than 70% were considered sufficiently resolved (
Pairwise comparisons of uncorrected p-distances for nine COI sequences (1,267 bp) obtained from specimens of the new species in this study and sequences from Orobdella masaakikuroiwai Nakano, 2014 and Orobdella naraharaetmagarum Nakano, 2016 were calculated using MEGA6.06 (
Orobdella Oka, 1895: 278–280;
Kumabdella
Orobdella whitmani Oka, 1895 by subsequent designation of
Body firm and muscular, elongate, with constant width in caudal direction, dorsoventrally compressed. Somite I completely merged with prostomium. Somite II uniannulate, not separated from I. Mid-body somite annulation variable, complete quadr-, sex- or octannulate. Post-anal annulus absent. Male gonopore in posterior part of XI. Female gonopore in anterior part of XIII. Pappilae numerous, minute, hardly visible, one row on every annulus. Pharynx agnathous, euthylaematous. Crop tubular, acaecate. Gastropore, when present, in anterior part of XIII. Gastroporal duct generally lying on female organ. Intestine tubular, acaecate. Rectum tubular, thin-walled, straight. Testisacs multiple. Ejaculatory bulbs absent. Male median reproductive system in posterior part of XI, without penis or penis sheath. Ovisacs globular. Oviducts thin-walled. Common oviduct thin-walled, short. Female median reproductive system essentially lacking.
Holotype:
Japan, Tokushima Prefecture: Mima, Mt. Ichinomori (Shikoku).
Body length of mature individual reaching to ca. 5 cm. Somite IV uniannulate, somites VIII–XXV quadrannulate. Clitellum in XI b5 to XIII a2. Male gonopore in middle of XI b6, female gonopore in anterior margin of or slightly anterior to middle of XIII a1, behind gastropore, gonopores separated by 1/2 + 4 [+ (< 1/2)] annuli. Pharynx reaching to XIV a1–a1/a2. Gastropore conspicuous, in anterior margin of or slightly anterior to middle of XIII a1. Gastroporal duct tubular, slightly bulbous at junction with gastropore. Paired epididymides in XX to XXI, occupying four annuli (one somite). Atrial cornua small ovate.
BL 51.6 mm, BW 5.2 mm (Fig.
Orobdella brachyepididymis sp. n., holotype,
Somites III, IV uniannulate; IV with slight dorsal furrow (Fig.
X b5 and XIII a2, respectively, being first and last annuli of clitellum (Fig.
Male gonopore in middle of XI b6 (Fig.
Anterior ganglionic mass in VI a2 and a3. Ganglion VII in a2. Ganglion VIII in a2 and b5. Ganglia IX and X, of each somite, in a2. Ganglion XI in a2 and b5 (Fig.
Orobdella brachyepididymis sp. n., holotype,
Eyes in three pairs, first pair dorsally on posterior margin of II, second and third pairs dorsolaterally on posterior margin of V (a1 + a2) (Fig.
Nephridiopores in 17 pairs, one each situated ventrally at posterior margin of a1 of each somite in VIII–XXIV (Fig.
Pharynx reaching to XIV a1 (Fig.
Testisacs (Fig.
Paired ovisacs in XIII a2 and b5 (Fig.
BL 36.3 mm, BW 3.5 mm, CL 1.8 mm, CW 2.2 mm. Somite IV uniannulate Somite VI triannulate, a1 = a2 = a3. Male gonopore in middle of XI b6, female gonopore in slightly anterior to middle of XIII a1, gonopores thus separated by 1/2 + 4 + (< 1/2) annuli. Pharynx reaching to XIV a1/a2. Crop reaching to XX a1/a2. Gastropore in slightly anterior to middle of XIII a1. Gastroporal duct tubular, but slightly bulbous at junction with gastropore, joining with crop in XIV a2/b5. Intestine reaching to XXIV a1/b5. Testisacs undetectable. Paired epididymides; right epididymis in XX b5 to XXI a2/b5; left epididymis in XX a2/b5 to XXI a2; each occupying four annuli. Atrium in XI b6. Left oviduct crossing ventrally beneath nerve cord.
The specific name is a compound noun in apposition derived from the Greek words transliterated into Latin, brachys (short) and epididymis (epididymis), referring to the fact that the epididymides of this species occupy only four annuli.
This species was found only from its type locality.
This species was found curled up under rocks in moist mountainous habitats. As oligochaete worms were observed in the digestive tract of a dissected specimen,
A mature leech,
The specimens were small (up to 52 mm), but the holotype was determined to be mature because it possessed an obvious clitellum and developed testisacs.
According to taxonomic studies (
Comparisons of morphological characters between Orobdella brachyepididymis sp. n. and eight quadrannulate congeneric species.
Character | Body length | Somite IV | Somite XXV | Gastroporal duct | Annuli between gonopores | Epididymides | Atrial cornua |
---|---|---|---|---|---|---|---|
Orobdella brachyepididymis sp. n. | less than or reaching to ca. 5 cm | uniannulate | quadrannulate | tubular | 1/2 + 4 [+ (< 1/2)] | XX to XXI | small, ovate |
Orobdella esulcata | reaching to ca. 10 cm | uniannulate | quadrannulate | tubular, but bulbous at junction with gastropore | 2/3 + 4 + 1/3 | XVI to XX | developed, ovate |
Orobdella kawakatsuorum | reaching to ca. 10 cm | biannulate | quadrannulate | simple tubular | 6 | XVI to XVII | undeveloped |
Orobdella ketagalan | reaching to ca. 10 cm | uniannulate | quadrannulate | simple tubular | 1/2 + 4 + 1/2 | absent | undeveloped |
Orobdella koikei | less than 4 cm | uniannulate | triannulate | bulbous | 1/2 + 4 + 1/2 | XV to XX | developed, ovate |
Orobdella masaakikuroiwai | less than 4 cm | uniannulate | quadrannulate | bulbous | 1/2 + 4 + 1/2 | XVI to XVIII | developed, ovate |
Orobdella naraharaetmagarum | less than 5 cm | uniannulate | quadrannulate | bulbous | 1/2 + 4 + 1/2 | XV to XX | developed, ellipsoid or ovate |
Orobdella tsushimensis | reaching to ca. 10 cm | uniannulate | quadrannulate | bulbous | 1/2 + 5 | XVII to XIX | developed, ovate |
Orobdella whitmani | reaching to ca. 10 cm | uni- or biannulate | quadrannulate | bulbous | 1/2 + 4 + 1/2 | XVI to XVIII | developed, ovate |
Holotype:
Japan, Kochi Prefecture: Ino, Ishizuchi Mountains, Mt. Takanosuyama (Shikoku).
Body length of mature individual greater than 15 cm. Somites IX–XXV octannulate. Clitellum in X c9 to XIII b4. Male gonopore in slightly posterior to middle of XI c11 or XI c10/c11, female gonopore in XIII b2/b3, behind gastropore, gonopores separated by 1/2 + 11 or 12 annuli. Pharynx reaching to XIV b1–b3/b4. Gastropore conspicuous, in XIII b2/b3. Gastroporal duct bulbous, slightly winding at junction with gastropore. Paired epididymides in XV to XVII, occupying 12 or 13 annuli (one and half somites). Atrial cornua ovate or ellipsoid.
BL 237.7 mm, BW 11.3 mm (Fig.
Somites III–V biannulate; III and V, (a1 + a2) = a3; IV, (a1 + a2) > a3; V a3 forming posterior margin of oral sucker (Fig.
X c9 and XIII b4, respectively, being first and last annuli of clitellum (Fig.
Male gonopore in slightly posterior to middle of XI c11 (Fig.
Anterior ganglionic mass in VI b5 and b6. Ganglion VII in a1. Ganglia VIII–X, of each somite, in b3 and b4. Ganglion XI in b4 (Fig.
Orobdella nakahamai sp. n., holotype,
Eyes undetectable.
Nephridiopores in 17 pairs, one each situated ventrally at posterior margin of b2 of each somite in VIII–XXIV (Fig.
Pharynx reaching to XIV b1 (Fig.
Testisacs (Fig.
Paired ovisacs in XIII b3 and b4 (Fig.
BL 162.3–180.4 mm, BW 7.6–98 mm, CL 3.3–4.3 mm, CW 5.6 mm. Somite VI quadrannulate, a1 (dorsally b1 = b2) = a2 (dorsally b3 = b4) > b5 < b6, or dorsally quinquannulate, b1 = b2 < a2 > b5 < b6. Somite VII quinquannulate, a1 (dorsally b1 = b2 in KUZ Z1680) > b3 = b4 = b5 = b6. Somite VIII octannulate, b1 = b2 < b3 < b4 = c9 = c10 = c11 = c12, or ventrally septannulate, a1 (b1 = b2) > b2 = b3 = b4 = c9 = c10 = c11 = c12. Somite IX octannulate, b1 = b2 = b3 = b4 = c9 = c10 = c11 = c12. Somite XXVI dorsally octannulate, b1 = b2 = b3 = b4 > c9 = c10 = c11 = c12, ventrally septannulate, b1 = b2 = b3 = b4 > c9 = c10 < b6, or sexannulate, b1 = b2 = b3 = b4 = b5 < b6 (c11 = c12). Somite XXVII comprises two annuli, first annulus with slight three dorsal furrows, or comprises four annuli. Eyes in one pair, dorsally on posterior margin of II (KUZ Z1352). Pharynx reaching to XIV b3/b4. Crop reaching to XXII b1/b2. Gastroporal duct joining with crop in XIV b3. Intestine reaching to XXV b4. Male gonopore in XI c10/c11 (KUZ Z1680), thus gonopores separated by 12 annuli. Testisacs in XVII c10 to XXV c11; on right side, in total approx. 122 testisacs, 5 in XVII, 18 in XVIII, 17 in XIX, 18 in XX, 16 in XXI, 15 in XXII, 14 in XXIII, 10 in XXIV, 9 in XXV; on left side, in total approx. 126 testisacs, 4 in XVII, 16 in XVIII, 19 in XIX, 21 in XX, 17 in XXI, 13 in XXII, 13 in XXIII 11 in XXIV, 12 in XXV. Paired epididymides in XV c10/c11 to XVII b3/b4, occupying 13 annuli. Pair of muscular atrial cornua ellipsoid. Left oviduct crossing ventrally beneath nerve cord; both oviducts converging into common oviduct in XIII b3.
The specific name is a noun in the genitive case formed directly from the name of Mr Naoyuki Nakahama, who collected a specimen of this new species.
This species was collected from the Ishizuchi Mountains and south-western part of Kochi Prefecture. The lowest elevation among the localities was ca. 140 m, and the highest was ca. 1600 m. The locality data for this species suggested that it is distributed in mountainous regions in the western part of Shikoku, Japan. Its distribution may not be restricted by habitat elevation.
Mature leeches with an obvious clitellum,
Orobdella nakahamai is only the second octannulate large-type species known within the genus. According to
Comparisons of morphological characters between octannulate Orobdella nakahamai sp. n. and Orobdella octonaria.
Character | Female gonopore | Annuli between gonopores | Epididymides |
---|---|---|---|
Orobdella nakahamai sp. n. | XIII b2/b3 | 1/2 + 11 or 12 | XV to XVII |
Orobdella octonaria | middle of XIII b2 | 1/2 + 10 + 1/2 | XVII to XIX |
Holotype:
Japan, Kochi Prefecture: Kochi, Tosayamatakakawa, Mt. Kuishiyama (Shikoku).
Dorsal surface reddish. Somite VII quinquannulate. Somite VIII–XXVI sexannulate. Male gonopore in slightly posterior to middle of XI c11/c12, female gonopore in middle of XIII b2, behind gastropore, gonopores separated by 8 + 1/2 annuli. Pharynx reaching to XIV b1–b2/a2. Gastropore conspicuous, in middle of XIII b2. Gastroporal duct bulbous, slightly winding at junction with gastropore. Paired epididymides in XV to XVII, occupying 8–11 annuli (one and half to almost two somites). Pre-atrial loop absent. Atrial cornua ellipsoid.
BL 95.2 mm, BW 5.8 mm (Fig.
Orobdella okanoi sp. n., holotype,
Somite III uniannulate (Fig.
Male gonopore in XI c11/c12 (Fig.
Anterior ganglionic mass in VI a1 and VII b1. Ganglion VII in b2 and a2. Ganglion VIII in a2. Ganglion IX in a2 and b5. Ganglion X in a2. Ganglion XI in a2 and b5 (Fig.
Orobdella okanoi sp. n., holotype,
Eyes in three pairs, first pair dorsally on posterior margin of II, second and third pairs dorsolaterally on posterior margin of V (a1 + a2) (Fig.
Nephridiopore in 17 pairs, one each situated ventrally at posterior margin of b2 of each somite in VIII–XXIV (Fig.
Pharynx reaching to XIV b1 (Fig.
Testisacs (Fig.
Paired ovisacs; right ovisac in XIII a2 and b5; left ovisac in XIII a2–c11 (Fig.
BL 143.2 mm, BW 7.6 mm, CL 3.1 mm, CW 4.0 mm. Somite III uniannulate with slight dorsal furrow. Somite XXVII comprises two annuli, each annulus with slight dorsal furrow. Male gonopore in posterior margin of XI c11. Female gonopore in middle of XIII b2. Eyes in three pairs, firs pair dorsally on anterior margin of III. Pharynx reaching to XIV b2/a2. Crop reaching to XXII a2. Gastropore in middle of XIII b2. Gastroporal duct joining with crop in XIV b2. Intestine reaching to XXIV c12. Testisacs; on right side, in XVII b2 to XXI b5, in total approx. 64 testisacs, 8 in XVII 13 in XVIII, 18 in XIX, 17 in XX, 8 in XXI; on left side, in XVII b2 to XXIV c11, in total approx. 99 testisacs, 7 in XVII, 10 in XVIII, 17 in XIX 17 in XX, 15 in XXI, 13 in XXII, 11 in XXIII, 9 in XXIV. Paired epididymides; right epididymis in XV a2 to XVI/XVII, occupying 10 annuli; left epididymis in XV a2 to XVII b1, occupying 11 annuli. Paired ejaculatory ducts curved in position anterior to ovisacs. Paired atrial cornua; right cornu in XI c12 and XII b1; left cornu in XI b5 and c11. Paired ovisacs in XIII a2 and b5.
In life, dorsal surface red-purple (Fig.
The specific name is a noun in the genitive case formed directly from the name of Mr Ryosuke Okano, who collected valuable specimens of Orobdella leeches.
The type locality of this species is located in the central region of Shikoku, Japan. In addition, this species was collected from the Ishizuchi Mountains. According to the collection localities, this species is considered to inhabit the central mountainous region of Shikoku.
This species was found curled up under a rock or in soil in moist mountainous habitats. Oligochaete worms were found in the digestive tract of the holotype, and thus this species is an earthworm-eater. The reproductive season of O. okanoi remains unclear because no individuals of this species with a clitellum have been collected.
According to taxonomic studies on sexannulate Orobdella species (
Comparisons of morphological characters between Orobdella okanoi sp. n., Orobdella yamaneae sp. n. and four sexannulate congeneric species.
Character | Dorsal colour | Somite VII | Somite VIII | Annuli between gonopores | Pharynx | Gastroporal duct | Epididymides | Pre-atrial loop | Atrial cornua |
---|---|---|---|---|---|---|---|---|---|
Orobdella okanoi sp. n. | reddish | quinquannulate | sexannulate | 8 + 1/2 | reaching to XIV | bulbous | XV to XVII | absent | ellipsoid |
Orobdella yamaneae sp. n. | purplish | quinquannulate | sexannulate | 1/2 + 7 + 1/2 | reaching to XIV | bulbous | XVI to XVIII | extending to anterior of XI c9 | ovate |
Orobdella dolichopharynx | yellowish green | quadrannulate | quinquannulate | 8 | reaching to XVI | tubular, reaching to XVI | absent | extending to ganglion XI | absent |
Orobdella ijimai | yellowish green | quadrannulate | sexannulate | 1/2 + 7 + 1/2 | reaching to XIV | bulbous | XVI to XIX | absent | ellipsoid |
Orobdella mononoke | anterior and posterior parts grayish purple, mid-body amber | quadrannulate | sexannulate | 8 + 1/2 | reaching to XIV | tubular, but bulbous at junction with crop | XV to XIX | absent | ovate |
Orobdella shimadae | yellowish green | triannulate | quinquannulate | 9 | reaching to XVI | tubular, reaching to XV | absent | extending to ganglion XI | absent |
The right atrial cornu of one specimen,
Holotype:
Japan, Hyogo Prefecture: Minamiawaji, Mt. Yuzuruhasan (Awajishima island).
Dorsal surface purplish. Somite VII quinquannulate. Somite VIII–XXVI sexannulate, b1 = b2 = a2 = c9 = c10 = b6. Male gonopore in middle of XI b6, female gonopore in slightly posterior to middle of XIII b2, behind gastropore, gonopores separated by 1/2 + 7 + 1/2 annuli. Pharynx reaching to XIV a2/c9–c9. Gastropore conspicuous, in slightly posterior to middle of XIII b2. Gastroporal duct bulbous, slightly winding at junction with gastropore. Paired epididymides in XVI to XVIII, occupying 8–11 annuli (one and half to almost two somites). Pre-atrial loop present. Atrial cornua ovate.
BL 142.2 mm, BW 8.1 mm (Fig.
Orobdella yamaneae sp. n., holotype,
Somites III–V biannulate; III and IV, (a1 + a2) > a3; V, (a1 + a2) = a3; V a3 forming posterior margin of oral sucker (Fig.
X c9 and XIII a2, respectively, being first and last annuli of clitellum (Fig.
Male gonopore in middle of XI b6 (Fig.
Anterior ganglionic mass in VI a2 and a3. Ganglion VII in a2. Ganglia VIII and IX, of each somite, in b2 and a2. Ganglia X–XII, of each somite, in a2 (Fig.
Orobdella yamaneae sp. n., holotype,
Eyes in three pairs, first pair dorsally on II/III, second and third pairs dorsolaterally on posterior margin of V (a1 + a2) (Fig.
Nephridiopores in 17 pairs, one each situated ventrally at posterior margin of b2 of each somite in VIII–XXIV (Fig.
Pharynx reaching to XIV a2/c9 (Fig.
Testisacs in XVIII c9 to XXVI b1 (Fig.
Paired ovisacs in XIII a2 and c9 (Fig.
BL 62.4–97.6 mm, BW 4.0–6.5 mm, CL 1.6–2.8 mm, CW 2.5–3.6 mm. Somite III uniannulate with slight dorsal furrow, [(a1 + a2) > a3]. Somite IV uniannulate with slight dorsal furrow, [(a1 + a2) > a3] in
In life, dorsal surface grayish purple or red-purple (Fig.
The specific name is a noun in the genitive case formed directly from the name of Ms Yoshiko Yamane, who collected specimens of this new species.
This species was collected only from its type locality.
This species was found curled up under fallen leaves in moist mountainous habitats. A mature leech,
The mid-body somite annulation of the known sexannulate Orobdella species was described as being composed of b1, b2, a2, b5, c11 and c12 (
In addition to its unique sexannulation, O. yamaneae is distinguishable from the four known sexannulate species and O. okanoi by the following characteristics (Table
Except for the holotype, all dissected individuals possess the following characteristics of the male genital organ: ejaculatory ducts in position anterior to ovisacs running straight and male atrial cornua ellipsoid or fusiform. However, they seem to be immature leeches because all of them have undeveloped and undetectable testisacs. Therefore, straight ejaculatory ducts and ellipsoid or fusiform testisacs are considered to be immature characteristics of O. yamaneae.
The obtained BI tree (Fig.
Bayesian inference tree for 5,209 bp of nuclear 18S rRNA and histone H3 and mitochondrial COI, tRNACys, tRNAMet, 12S rRNA, tRNAVal, 16S rRNA, tRNALeu and ND1 markers. Numbers on nodes represent bootstrap values for maximum likelihood and Bayesian posterior probabilities. A species name of Orobdella in red indicates a quadrannulate species; green, a sexannulate; and blue, an octannulate species. Locality numbers are shown in Fig.
Orobdella brachyepididymis formed a monophyletic clade with O. naraharaetmagarum (BS = 100%, BPP = 1.0). This clade is a sister lineage to O. esulcata (BS = 100%, BPP = 1.0). The monophyly of the two specimens identified as O. brachyepididymis was fully supported (BS = 100%, BPP = 1.0). The other three new species, O. nakahamai, O. okanoi and O. yamaneae formed a monophyletic clade (BS = 100%, BPP = 1.0). The monophyly of this clade and O. masaakikuroiwai was revealed, but this relationship was not strongly supported in the BI tree (BS = 87%, BPP = 0.90). Within the three new species, O. okanoi and O. yamaneae formed a monophyletic lineage (BS = 78%, BPP = 0.94). The monophyly of the specimens of the three new species was fully recovered (O. nakahamai, BS = 98%, BPP = 1.0; O. okanoi, and O. yamaneae, respectively, BS = 100%, BPP = 1.0).
The pairwise COI uncorrected p-distances within each of the new species were as follows: in O. brachyepididymis, 0.5%; O. nakahamai, 2.1–4.6% (mean = 3.7%); O. okanoi, 2.4%; and O. yamaneae, 3.9% (Table
Uncorrected p-distances for the 1267 bp for the COI sequences of specimens of Orobdella nakahamai sp. n., Orobdella okanoi sp. n. and Orobdella yamaneae sp. n., with associated collection locality numbers (see Fig.
Species | Specimen (locality #) | 1 | 2 | 3 | 4 | 5 | 6 |
---|---|---|---|---|---|---|---|
Orobdella nakahamai sp. n. | 1: |
||||||
2: |
0.021 | ||||||
3: |
0.043 | 0.046 | |||||
Orobdella okanoi sp. n. | 4: |
0.058 | 0.056 | 0.056 | |||
5: |
0.062 | 0.058 | 0.058 | 0.024 | |||
Orobdella yamaneae sp. n. | 6: |
0.056 | 0.057 | 0.058 | 0.059 | 0.059 | |
7: |
0.057 | 0.056 | 0.058 | 0.058 | 0.060 | 0.004 |
According to the morphological characteristics of the four new species, each of them can be well defined and distinguished from each other and from the previously known species of Orobdella. However, the genetic divergences of the COI sequences showed small interspecific divergences. The genetic distance between Orobdella brachyepididymis and its sister species O. naraharaetmagarum (4.7%) was equivalent to the largest intraspecific COI divergence of the latter species indicated by
Calculated interspecific COI distances among the three other new species, O. nakahamai, O. okanoi and O. yamaneae, each of which is well defined by morphological characteristics, were also small. These values and the obtained phylogenetic trees indicated that these three species are closely related to each other. Orobdella nakahamai is sympatric with O. okanoi on Mt. Iwagurosan in the Ishizuchi Mountains (locality #5, see Fig.
According to the obtained molecular phylogenies, mid-body somite annulation and body size of mature leeches within Orobdella clearly evolved in parallel.
The sexannulation of O. yamaneae and the fact that this species is genetically quite close to O. okanoi imply the possibility that the sexannulation of O. okanoi also follows b1 = b2 = a2 = c9 = c10 = b6, and not the b1 = b2 = a2 = b5 = c11 = c12 observed in the other known sexannulate species of Orobdella. The annulation pattern of O. yamaneae suggests that annular formulae can vary among Orobdella species possessing the same annulation. The quadrannulate somite of Orobdella obviously consists of a1, a2, b5 and b6, according to the positions of paired nephridiopores and the ventral ganglion in each somite. However, the annulation pattern of sexannulate and octannulate Orobdella species should be determined based on several specimens, including immature individuals.
The present phylogenetic tree showed that 17 Orobdella species consist of four main lineages: a Hokkaido lineage containing two species, O. kawakatsuorum and O. koikei; anOrobdella tsushimensis lineage; a western lineage comprising four species inhabiting the Ryukyu Islands and Taiwan, along with O. esulcata distributed in Kyushu, O. naraharaetmagarum in the Chugoku district, western Honshu, as well as the new species O. brachyepididymis from Shikoku; and an eastern lineage comprising four species known from the eastern to central parts of Honshu, along with the other three new species, O. nakahamai and O. okanoi collected from Shikoku and O. yamaneae from Awajishima island. Therefore, the range of the western lineage group overlaps that of the eastern lineage group in Shikoku and adjacent islets. Because species belonging to both lineage groups are distributed in Shikoku, the species diversity of Orobdella in this region may be quite high compared to other regions. The new Orobdella species inhabiting Shikoku and Awajishima island would offer a suitable opportunity to reveal speciation events, as well as species coexistence mechanisms in the genus Orobdella.
The author is grateful to Naoyuki Nakahama (Kyoto University; KU), Ryosuke Okano (Ehime University), and Yoshiko Yamane (KU) for providing specimens of the new species, to Professor Hidetoshi Nagamasu (The Kyoto UniversityMuseum) for his helpful advice on a specific name of the new species. The author expresses his sincere thanks to Dr Yi-Te Lai (National Taiwan University) and Dr Michael Ohl (Museum für Naturkunde) 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 KU, and JSPS Grants-in-Aid for JSPS Fellows (#15J00720) and Young Scientists (B) (#26840127) to the author. The open access publication of this manuscript was supported by the Museum für Naturkunde.