New earthworm species of the genus Amynthas Kinberg, 1867 from Thailand (Clitellata, Oligochaeta, Megascolecidae)

Abstract Four new species of terrestrial earthworms from the zebrus-group in the genus Amynthas Kinberg, 1867, are described from Nan province, north Thailand: Amynthas phatubensis sp. n., from Tham Pha Tub Arboretum, Amynthas tontong sp. n., from Tontong Waterfall, Amynthas borealis sp. n., from Chaloemprakiat district, and Amynthas srinan sp. n., from Srinan National Park.After comparing with the two closely related Laos species Amynthas chandyi Hong, 2008 and Amynthas namphouinensis Hong, 2008, the four new species show clear morphological differences, and also it is confirmed that there are no previous records of the species described here. Amynthas phatubensis sp. n. is the largest (longest) sized of these earthworms and is the only species that lives in limestone habitats. The genital characters are different among them and also from the two Laotian species. Molecular systematics would be a good method for further analysis of the diversity and species boundaries in SE Asian Amynthas.

. Morphological characteristics comparison of Amynthas species recorded in Th ailand. Th e morphological characters are from the original description of each nominal species, except for the character with (*) are from Gates (1972). (**) indicate the known localities of Amynthas species in Th ailand taken from Gates (1972), Kosavititkul (2005) and Somniyam (2008). Species group are as per Sims and Easton (1972) Species  (Stephenson, 1931) Figure 1. Map of type locality of 1 Amynthas srinan sp. n. from Srinan National Park, Nan province, 2 Amynthas phatubensis sp. n. from Th am Pha Tub Arboretum, Nan province, 3 Amynthas tontong sp. n. from Tontong Waterfall, Pua district, Nan province and 4 Amynthas borealis sp. n. from a small hill near Chaloemprakiat district, Nan province.

Material and methods
Earthworms were collected from deciduous forests in many areas in Nan province, north of Th ailand, by carefully digging up the topsoil near casts and by hand sorting the leaf litter. Th e worms were killed in 30% (v/v) ethanol, photographed, transferred to 5% (w/v) formalin for fi xation for approximately 12 hours, and then transferred to 70% (v/v) ethanol for longer term preservation and subsequent morphological studies. Duplicate specimens and/or tissue samples (in the cases of morphotypes determined to be unique on fi eld inspection) were preserved in 95% ethanol for molecular data and DNA barcoding. Tissues were sent to the Canadian Center for DNA Barcoding (Hebert et al. 2003a, b) and processed according to their standard protocols (Hajibabaei et al. 2005;Ivanova et al. 2006;Ratnasingham and Hebert 2007). DNA barcode data are provided for paratype specimens of the fi rst two species described in this paper. Th e sequences were aligned with Clustal X using default settings, and the resulting Neighbor-Joining tree (Saitou and Nei 1987) was used to identify barcode clusters. Th ese clusters were matched to OTUs identifi ed from quick examination of external characters. Inter-and intra-cluster genetic distances were calculated in MEGA 4 (Tamura et al. 2007) using the Kimura two parameter distance (Kimura 1980) using gamma-distributed rates among sites, pairwise deletion of sites with missing data, and using all substitution types and codon positions.
Th e descriptions of each species were made during observation under a Stemi DV 4 ZEISS stereoscopic light microscope. Drawings were made of the body segments and the distinct external characters and internal organs, as mentioned above, and are shown in Figures 2-5 for the four new species, respectively. Th e number of segments and the body width and length were measured in both full adults and juveniles, and are presented as the range (min-max) and mean±one standard deviation.
Type specimens housed at the Department of Biology, Faculty of Science, National University of Laos, Vientiane, Laos (BDNUL), of the two closely related Laos species, A. chandyi Hong, 2008 andA. namphouinensis Hong, 2008, have been critically studied and compared with the new species of this report.
Holotype and paratype specimens have been deposited in the Chulalongkorn University, Museum of Zoology, Bangkok, Th ailand (CUMZ). Additional paratypes are housed in the Biozentrum Grindel und Zoologisches Museum, Hamburg, Germany (UHH), and the Natural History Museum, London (NHM).
Anatomical abbreviations: fp, female pore; ic, intestinal caeca; mp, male pores; pg, prostate gland; sc, spermathecae; sp, spermathecal pores; sv, seminal vesicles. A pair of mp is located ventro-laterally in XVIII, or at 9 th seta line, 0.33 circumference apart ventrally, convex structure; distance between mp 4.2 mm. Porophores (protuberances bearing male aperture), papilla-like structures. Each mp surrounded by six fl at, circular genital markings almost the same diameter as mp, also one pair is equatorial in XVII in line with the male pores. One pair of sp in intersegmental furrow 7/8, distance between pores 0.32 circumference ventrally apart; distance between sp 3.5 mm. Genital markings, rounded, fl at, located close to sp, postsetal paired on VII very near 7/8, presetal paired on VIII.
Ovaries in XIII. Sc one pair in VIII; ampulla large ovate sac, duct stout, short; long stalked diverticulum, convoluted kinks enclosed within membrane, spherical knob terminal. No nephridia on spermathecal ducts. A large sessile genital marking gland corresponding to each external genital marking in VII-VIII. All the key morphological characters of the holotype and paratype specimens are given in Table 2.
Etymology: Th is species was named after the type locality, Th am Pha Tub Arboretum.
Habitat: Found in the top soil at about 10 cm depth, the soil surface was covered with leaf litter in a deciduous limestone forest at Th am Pha Tub Arboretum. Th e soil was carefully dug close to the casts. Many ariophantid snails, Cryptozona siamensis Pfeiff er, 1856 were on the ground or under leaf litter.
Diagnosis: Amynthas phatubensis sp. n. is a medium to large sized terrestrial earthworm with a pair of mp surrounded by six genital papillae on segment XVIII. Within the zebrus-group, this species is diagnosed by the unique combination of dorsal pores in 5/6, simple digitate caeca, ventrally joined testis sacs, genital marking glands in the spermathecal segments, and the spermathecal characters of the large ovate ampulla, stalked diverticulum whose folds are membrane-bound, and spherical knob terminal diverticulum sac.
Remarks: Amynthas phatubensis sp. n. has very simple characteristics of the genus, but among these, only the superfi cial male pores are external. In most newly collected specimens it was diffi cult to observe the pores or marks on the bodies. However, after preservation they can be seen more clearly. Th e internal organs are much more easily discerned. Th is new species is quite distinct when compared to the two closely related species from Laos, A. chandyi Hong, 2008 andA. namphouinensis Hong, 2008, which belong in the same zebrus-group. Th e two Laos species are a little bit smaller than A. phatubensis sp. n., especially A. chandyi. Even though A. namphouinensis is much closer in appearance to A. phatubensis sp. n., there are distinct diff erences between the type specimens (Figs 6 and 7). For example, the distance between the mp of A. phatubensis sp. n. is 4.2 mm for the holotype and range from 3.0-4.5 mm (4.27±0.57mm), while for A. namphouinensis this was signifi cantly smaller, ranging from 1.4-1.5 mm. Th e distance between a pair of sp is also diff erent, being 3.5-4.5 mm (4.12±0.4 mm) for A. phatubensis sp. n. and 1.4-2.0 mm in A. namphouinensis. Th e distance between the male pores as a fraction of the estimated circumference of the 18 th segment is 0.30-0.33 in A. phatubensis sp. n., but 0.10-0.14 circumference apart in A. namphouinensis. Two populations of A. phatubensis sp. n. were sampled, one from the type locality and one from Tontong waterfall. Distinct DNA barcode clusters corresponding to these populations had intra-cluster Kimura 2 parameter distances of 0.023 (N=9) and 0.016 (N=5) respectively. Th e inter-cluster divergence between the two populations is 0.084. Based on the morphological unity and the fact that the divergence is less than that usually seen between congeneric species pairs of earthworms (Chang et al. 2007;Pérez-Losada et al. 2005, James et al. 2010, we choose to maintain the two populations as representing one species. By contrast, the inter-cluster divergence between these populations and three other morpho-species with the same spermathecal battery, from the same two sites is in the range of 0.269-0.294. A consensus sequence from the type locality specimens is in Appendix 1. Another use of COI barcode sequence from type material is in Blakemore et al. (2010).
A pair of indistinct rounded mp in XVIII, 0.19 mm circumference apart ventrally; distance between mp 1.0 mm at 5 th seta line. Genital markings closely paired located medial to male pore level in intersegment 18/19. Sp paired in 7/8 at 4 th seta line, each small, lip-like structure within porophore, 0.10 circumference apart ventrally; distance between sp 1.0 mm. Genital markings near sp absent.
Ovaries in XIII. Sc one pair in VIII; ampulla thumb shape, duct stout, shorter than ampulla. Diverticulum slender stalk with spherical knob terminal, no genital marking glands observed.
All the key morphological characters of the holotype and paratype specimens are given in Table 3.
Etymology: Th is species was named after the type locality, Tontong Waterfall. Type material: Th e holotype (CUMZ 3206) and two paratypes (CUMZ 3207) are deposited in Chulalongkorn University, Museum of Zoology. Another paratype will be deposited in the Biozentrum Grindel und Zoologisches Museum, Hamburg, Germany (UHH).
Habitat: Found in the top soil at about 10 cm depth, the soil surface covered with leaf litter of deciduous forest which originated at the Tontong Waterfall area. Th e soil was carefully dug close to surface casts. Most surrounding areas have been modifi ed to agricultural fi elds.

Diagnosis:
Amynthas tontong sp. n. is a small sized terrestrial earthworm with a close indistinct pair of male pores with a pair of genital markings in intersegment 18/19. Spermathecae consists of a thumb shaped ampulla and a spherical terminal knob shaped diverticulum. Genital marking glands absent, fi rst dorsal pore in 5/6, intestinal caeca simple, intestinal origin XV, septa 8/9/10 absent, testis sacs joined ventrally.
Remarks: Amynthas tontong sp. n., along with A. srinan sp. n. and A. exiguus exiguus, is one of the smallest sized Amynthas ever recorded in Th ailand. Th e basic external characters are easily seen in both newly collected and preserved materials. Compared with the two other closely related species from Laos, A. chandyi Hong, 2008 andA. namphouinensis Hong, 2008, which belong in the same zebrus-group, A. chandyi is similar to A. tontong sp. n. However, it diff ers in the specifi c details of the signifi cant characters, such as the distance between the mp in A. tontong sp. n. is 1.0 mm for the holotype and ranged from 1.0-1.2 mm (0.93±0.12 mm), while in A. chandyi it ranged from 1.5-2.4 mm. Th e distance between the male pores as a fraction of the estimated circumference of the 18 th segment is 0.15-0.19 in A. tontong sp. n., but 0.14-0.32 in A. chandyi. Th e arrangement of the genital markings of both species are totally diff erent, and the distance between a pair of sp is also diff erent, being 0.8-1.0 mm (1.1±0.1 mm) in A. tontong sp. n. and 1.2-1.5 mm for A. chandyi. Moreover, A. tontong sp. n. has no genital markings near to the sp, whilst A. chandyi exhibits circular genital markings in various locations, paired or single mid ventral in VII, VIII; usually 3 or 4 in total.
Alcohol-preserved paratype specimens of A. tontong sp. n. belonged to a single DNA barcode cluster, with an intra-cluster divergence of 0.005 (N=3), and diverging from A. phatubensis sp. n. by 0.294, and by 0.189 for an undescribed species. An undescribed morph at Th am Pha Tub diverged by 0.100, and may represent a subspecies. A consensus sequence is in Appendix 1. Description of Holotype: Dimensions; 54 mm by 3.5 mm at segment X, 3.8 at segment XX, 3.5 mm at clitellum; body cylindrical with 89 segments. Setae regularly distributed around segmental equators, numbering 39 at VII, 51 at XX, no visible setae between mp, setae formula AA:AB:ZZ:ZY= 2:1:1.5:1 at XIII. Single fp at XIV. Prostomium epilobic. First dorsal pore at 5/6. Clitellum annular XIV-XVI with no setae.

Amynthas borealis
Mp pocket-like structures indistinctly occur in XVIII, 0.10 circumference apart ventrally; distance between mp 1.0 mm; porophores small, lip-like and surrounded by an elevated skin fold at medial pores, and there is a long ridge with a sharp posterior boundary traversing the body in front of the mp. Genital markings absent. Sp paired in 7/8 at 4 th seta line, 0.10 circumference apart ventral; distance between sp 1.0 mm. Genital markings absent.
Ovaries at XIII. Sc one pair in VIII; ampulla large sac-shape, fl attened by gizzard, narrow duct shorter than ampulla. Diverticulum with elongated tubular shape, stalk attached to duct near body wall, with no genital marking glands.
All the key morphological characters of the holotype and paratype specimens are given in Table 4.
Etymology: Th e specifi c epithet "borealis" derived from Latin word "boreal" mean "north". Th is name refers to the location of type locality in the north of Th ailand.
Type material: Th e holotype (CUMZ 3208) and seven paratypes (CUMZ 3209) are deposited in Chulalongkorn University, Museum of Zoology. Another two paratypes will be deposited in the Biozentrum Grindel und Zoologisches Museum, Hamburg, Germany (UHH), and another two paratypes in the Natural History Museum, London (NHM).
Habitat: Found in the top soil at about 10 cm depth, the soil surface covered with the leaf litter of a deciduous limestone forest, mostly disturbed. Th e soil was carefully dug close to the casts.
Diagnosis: Amynthas borealis sp. n. is a small sized terrestrial earthworm small male pores, a transverse ridge anterior to the male pores in XVII, and no genital mark-ings. One pair of sc in VIII, each spermathecae consists of a large sac-shaped ampulla and elongated tubular shaped diverticulum. Testis sacs joined ventrally, intestinal origin XV, intestinal caeca simple, fi rst dorsal pore in 5/6. Remarks: Amynthas borealis sp. n. is one of the smaller Amynthas. Th e characteristic male fi eld is diffi cult to see in newly collected specimens but can be clearly observed after preservation. Compared with the two other closely related species from Laos, A. chandyi and A. namphouinensis, which belong in the same zebrus-group, A. chandyi is similar to A. borealis sp. n. However, distinctive diff erences include the distance between mp of the new species, being 1.0 mm in the holotype with a range of 0.8-1.0 mm (0.95±0.09 mm) in A. borealis sp. n. compared to 1.5-2.4 mm. Th e distance between the male pores as a fraction of the estimated circumference of the 18 th segment is 0.10-0.14 in A. borealis sp. n., but 0.14-0.32 in A. chandyi. Th ere are no genital markings in the new species; the distance between a pair of sp is also diff erent, being 0.5-1.0 mm (0.9±0.19 mm) in the new species compared to 1.2-1.5 mm for A. chan- dyi. Moreover, A. borealis sp. n. has no genital marking glands at all, whilst A. chandyi exhibits circular genital markings in various locations, paired or single mid ventral in VII and VIII; usually 3 or 4 in total. Mp on circular porophores in XVIII, 0.30 circumference apart ventrally; distance between mp 1.5 mm. Genital markings small, postsetal, closely paired near mid ventral of XVII and XVIII. Sp paired in 7/8 at 6 th setal lines, 0.26 circumference apart ventrally; distance between sp 1.5 mm. Genital markings tiny, closely paired on near mid ventral of VII and VIII. Septa 5/6 and 6/7 thick, 7/8 thin, 8/9 and 9/10 absent, 10/11-13/14 thin. Gizzard globular within VIII-X, intestinal origin in XV, no lymph glands observed. Typhlosole small from XXVII. Ic originated from XXVII extending forward to XXIII, long fi nger-shape. Hearts esophageal in X-XIII. Holandric; testes and funnels in ventrally joined sacs in X-XI. Sv paired in XI-XII. Prostates in XVIII, extending between XVII-XX; prostatic ducts tightly folded twice. Genital marking glands paired in XVII and XVIII corresponding to external genital papillae, each consisting of a stalk with terminal multi-lobed glandular part.

Amynthas srinan
Ovaries in XIII. Sc one pair in VIII; ampulla oval to kidney-shaped, with stout duct shorter than ampulla. Diverticulum with oval bulb terminal, stalk attached to duct near body wall. Genital markings stalked, corresponding to external genital papillae; each gland small consisting of a stalk with terminal multi-lobed glandular part.  All the key morphological characters of the holotype and paratype specimens are given in Table 5.
Etymology: Th is species was named after the type locality Srinan National Park. Type material: Th e holotype (CUMZ 3210) and 25 paratypes (CUMZ 3211) are deposited in Chulalongkorn University, Museum of Zoology. Another fi ve paratypes will be deposited in the Biozentrum Grindel und Zoologisches Museum, Hamburg, Germany (UHH), and four paratypes in the Natural History Museum, London (NHM).
Habitat: Found in the top soil at about 10 cm depth, the soil surface covered with leaf litters of deciduous forest. Th e soil was carefully dug close to the castes.
Diagnosis: Amynthas srinan sp. n. is the smallest Amynthas ever collected in Th ailand. Male pores on distinct round porophores, genital markings paired near mid ventral of VII, VIII, XVII and XVIII; each with genital marking glands. Each spermathecae consists of a kidney-shaped ampulla and an oval shaped diverticulum. Testes sacs ventrally joined, intestinal origin XV, intestinal caeca simple, fi rst dorsal pores at 4/5 or 5/6. Remarks: Amynthas srinan sp. n., along with A. exiguus exiguus and A. tontong sp. n., is one of if not the smallest Amynthas recorded so far. It has external characteristics which are easily seen in both newly collected and preserved materials. Compared with the two other closely related species from Laos, A. chandyi and A. namphouinensis, which belong in the same zebrus-group, A. chandyi is very similar in appearance to A. srinan sp. n. However, they clearly diff er in certain specifi c details of their signifi cant characters, such as the distance between the mp which in A. srinan sp. n. is 1.5 mm for holotype and ranged from 1.5-2.0 mm (1.41±4.27 mm), while in A. chandyi this ranged from 1.5-2.4 mm. Th e distance between the male pores as a fraction of the estimated circumference of the 18 th segment is 0.24-0.30 in A. srinan sp. n., and 0.14-0.32 in A. chandyi. Th is is not convincing as a diagnostic diff erence, because there is signifi cant overlap with the highly variable A. chandyi. In addition, although genital markings are clearly observed in both A. chandyi and A. srinan sp. n. on the sc and mp areas, A. srinan sp. n. has a much larger number and diff erent arrangement of such markings. Th e distance between pairs of sp is quite similar, being 1.5-2.0 mm (1.34±2.31mm) in A. srinan sp. n. and 1.2-1.5 mm in A. chandyi.

Discussion
Th e genus Amynthas is widely distributed in the Asian continent, where it is one of the dominant genera. In Th ailand it occurs in various types of lowland forest habi-tats, dry evergreen, moist evergreen, deciduous and limestone forests, encompassing diverse soil pH values, from acidic to alkali soils (Chantaravisoot, 2007) and from clay to muddy sand substrates (Kosavititkul, 2005;Somniyam, 2008;Blakemore et al., 2007). Th e current four new species described here were all are found in one area (Nan province) but the four habitat types were quite diverse all the same. Amynthas phatubensis sp. n. was found in a limestone area with a mild alkali substrate (pH 7.5-8) of a clay loam structure, whilst the other three species were found in harder sandy clay substrates. Th e four new species are broadly similar (and so potentially related) to the two species described from Laos, A. chandyi and A. namphouinensis, but diff er in both the external and internal morphological characteristics. Th e geographic structures of Luang Prabang Mountain and Phi Pan Nam Mountain ranges are important barriers for species from both the Th ai (Nan province) and Laos side (Xayabouli province) and may have played an important part in their speciation. In addition, the Laos species live at a higher altitude than the current new described species from Th ailand, and such selective adaptations may facilitate their morphological discrimination.
Th e four new species range in size, with respect to other Amynthas members, from moderate to very small, of which A. phatubensis sp. n. is the longest. Th e other three species are almost the same size and close to the two Laotian species, as shown in Table 6. However, the spermathecae (sc) and genital marking locations of the four new species are clearly diff erent from the two closely related Laos species. Th e four new Amynthas species described here belong to the zebrus-group, as defi ned by Sims and Easton (1972),in which the spermathecal pores are located on segment 7/8. Th e size of these four species, relative to other Amynthas species, varied from small to medium, ranging from 35 to 148 mm in body length and having from 52 to 114 segments. Th e fi rst dorsal pore in three of the four species described here, and most of the samples of the fourth species (A. srinan sp. n.), is located on intersegmental furrow 5/6, but with some samples of A. srinan sp. n. showing the fi rst dorsal pore at 4/5.
Amynthas phatubensis sp. n. is the only species that lives in limestone habitats in leaf litter and also in shallow mild alkali topsoil. Th e soil humidity can be quite low and is of a clay loam structure. Th e other three species are smaller in size and were found in almost harder, muddy sandy clay substrates. Amynthas tontong sp. n. lives in deeper soil of a high humidity around waterfalls. Amynthas borealis sp. n. and A. srinan sp. n. are found in deciduous forests, which have mostly been modifi ed as agricultural fi elds. Th e soil is drier and harder. Th e genital marking glands of A. phatubensis sp. n. and A. srinan sp. n. are distinct from other two species (Table 6 and   Wang & Wang, 1993), A. heaneyi James, 2004, A. namphouinensis Hong, 2008and A. chandyi Hong, 2008. Within the zebrus-group, the fi rst three species show manicate intestinal caeca, while the current newly described four species have simple fi ngershaped intestinal caeca. Th e three latter nominal species are longer in body length (200-300 mm) compared with the size of these four new species which ranged from 35-148 mm. Amynthas heaneyi can be distinguished by its proandric character (James, 2004), while the four new described species are holandric. Amynthas fasciculus has coiled and kinked spermathecae, whereas A. phatubensis sp. n. has large ovate ampulla, A. tontong sp. n. has thumb shaped ampulla, A. borealis sp. n. has sac-shape ampulla, and A. srinan sp. n. has oval to kidney-shaped ampulla. Amynthas xuongmontis clearly diff ers from these four new species in the genital marking located on XVIII, whereas located on VII, VIII, XVII, XVIII in A. phatubensis sp. n., located between 18/19 in A. tontong sp. n., absent in A. borealis sp. n. and located on VII, VIII, XVII, XVIII in A. srinan sp. n.