New species of semi-aquatic freshwater earthworm genus Glyphidrilus Horst, 1889 from Thailand and Laos (Oligochaeta, Almidae)

Abstract Seven new species of semi-aquatic freshwater earthworms belonging to the genus Glyphidrilus Horst, 1889 are described from Thailand and Laos, Glyphidrilus nanensis Chanabun & Panha sp. n., G. satunensis Chanabun & Panha sp. n., G. chiangraiensis Chanabun & Panha sp. n., G. namphao Chanabun & Panha sp. n., G. sekongensis Chanabun & Panha sp. n., G. namdonensis Chanabun & Panha sp. n., and G. champasakensis Chanabun & Panha sp. n. The species are characterized by their external and internal morphological characteristics, as well as body sizes. Other morphological character differences between these seven species were also compared, and an identification key is provided. The relationships of the new species to congeners are discussed.


Introduction
The semi-aquatic freshwater earthworm genus Glyphidrilus Horst, 1889 has been known since the nineteenth century. The unique morphological characters of the expanded epidermis at approximately the clitellum position, called "wings", and the absence of prostate glands, the rounded body with a posterior quadrangular shape and the long slender banana-like cocoons are prominent in Glyphidrilus. The semiaquatic habitat between terrestrial and freshwater ecosystems of rivers, streams, canals, ponds, swamps or even in paddy rice systems are also consistent (Horst 1889, 1893, Jamieson 1968, Michaelsen 1896, 1897, 1900, 1902, 1910, 1918, Rao 1922, Shen and Yeo 2005, Chanabun et al. 2013. Up to now, the records of Glyphidrilus are only from Africa and Asia; however, most described species are from Asia and especially from Southeast Asia. The worms are now becoming threatened because of the modification, pollution, and destruction of their habitats, for example the contamination by chemical agriculture, and the dam constructions in the upper Mekong River (pers. obs.).
Most previous species were described with some illustrations in a format which was frequently poorly interpreted and insufficient in some species. However, in the recent descriptions and redescriptions of several species are mostly from Thailand, and some from Malaysia, Singapore, and Laos, these deficiencies have been corrected. The color images of both animals and habitats together with anatomical illustration details have made improvements in a new description format. The 19 newly described species reported bring the total number to 40 recognized Glyphidrilus species (Chanabun et al. 2011, Chanabun et al. 2012a, Chanabun et al. 2012b, Chanabun et al. 2013, Jirapatrasilp et al. 2016. The behavior of animals has been observed, and it was noted that the worms leave their tail tips exposed near surface of their muddy habitats. While submerging they produce casts as do most earthworms (Chanabun et al. 2013). It was also found that north of 12 degrees latitude in Thailand Glyphidrilus appears as mostly adults in the dry to early rainy seasons (March to July), while in the rainy season they mainly appeared as juveniles, especially in the upper parts of Thailand. However, south of 12 degrees latitude, the adults seem to be present all year round (pers. obs.). Chanabun et al. (2013) interpreted the phylogeography of Glyphidrilus in light of recent hypotheses regarding ancient river drainage patterns, especially the Mekong River and other main basins, plus various habitat types, using morphological characters and genetic data. Enzyme electrophoresis has proved that the closely related species G. mekongensis Chanabun, 2012 andG. vangviengensis Panha &Chanabun, 2011 occurring along the lower Mekong River basin are definitely separate biological species; some possible cryptic species are also suggested in the paper (Jirapatrasilp et al. 2015). The present paper provides additional new species with careful morphological descriptions but no additional genetic data.

Materials and methods
The systematic and faunistic surveys of Glyphidrilus were conducted in the lower Mekong River basin both in Thailand and Laos (Fig. 1), and some other river systems in Thailand (Fig. 2) from June 2012 to April 2014. The collections were made by carefully digging up the topsoil near casts on the shore and in the water using hand sorting and sieving the soil from river banks. Adults, juveniles, and cocoons were collected and killed in 30% (v/v) ethanol, transferred to 5% (w/v) formalin for fixation in approximately 12 hours, and then transferred to 75% (v/v) ethanol for standard preservation and subsequent morphological studies. Duplicate specimens and/or tissue samples were preserved in 95% ethanol for further molecular and DNA barcoding analyses.
The type series are deposited in the Chulalongkorn University, Museum of Zoology, Bangkok, Thailand (CUMZ). Additional paratypes will be deposited in the Natural History Museum (NHMUK), London, Raffles Museum of Biodiversity Research, National University of Singapore, Singapore (ZRC) and at the Biozentrum Grindel und Zoologisches Museum, University of Hamburg (ZMH).
The descriptions were made from observations under an Olympus SZX16 stereoscopic light microscope. The following external and internal morphological characters were investigated and recorded: body length and segment number; the positions of clitellum and clitellar wings, genital markings, intestinal origin, gizzard, spermathecae, hearts and seminal vesicles. Illustrations were made for the whole body segments and the external and internal characters. 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 (SD).
Comparative studies of Glyphidrilus type specimens were made at four natural history museums:

CUMZ
Chulalongkorn University, Museum of Zoology.

ZRC
Raffles Museum of Biodiversity Research, National University of Singapore, Singapore. NHMUK The Natural History Museum, London.
Diagnosis. A small sized earthworm with the clitellar wings on the lateral side of the body in XXIV, XXV, XXVI-XXVII, XXVIII, XXIX. Clitellum in XVII, XVI-II-XXXIII, XXXIV. Female pores, male pores, and spermathecal pores not visible. Genital markings: paired or asymmetrical on aa in X, XI, XII, XIII, XIV and XXVIII, XXIX, XXX; paired or asymmetrical on bc in XV, XVI, XVII-XXIII, XXIV, XXV. Four pairs of seminal vesicles in IX-XII. Intestinal origin in XV. Ovaries in XIII-XIV. Spermathecae between 13/14-17/18. Description of holotype. Dimensions: body length 78 mm, diameter 2.6 mm in segment VIII, 4.7 mm before the clitellar wing in segment XXIII, 4.2 mm after wing in segment XXX in clitellar region; body cylindrical in anterior part, quadrangular in transverse section behind clitellum. 241 segments. Body color pale brown with variations from red to pink at adjacent tissues of wing portion in different individuals of newly collected specimens. At posterior end dorsal surface considerably broader than the ventral. Clitellar wing on ventro-lateral part of clitellum in XXVI-XXIX, 3.6 mm in height, and 0.3 mm in width on both sides. Prostomium zygolobous. Dorsal pores absent. Clitellum annular in XVII-XXXIII. Four pairs of setae per segment from II, setal formula aa:ab:bc:cd:dd = 1.0:0.6:1.3:0.6:1.4 in segment VIII and 1.0:0.6:1.5:0.6:1.5 in postclitellar segments. Female pores, male pores, and spermathecal pores not visible. Genital markings: paired on aa in XII-XIII, laterally paired or asymmetrical on bc in XVII-XXIV. Septa 5/6-8/9 thicker than between other segments, 9/10-14/15 thick and 15/16 to the last segment thin. Gizzard small, globular in 7/8. Intestine enlarged from XV. Hearts, five pairs in VII-XI, first in VII and last in XI. A pair of holonephridia in each segment from segment XIII onwards. Seminal vesicles, four pairs in IX, X, XI, XII. Ovaries, two pairs in XIII-XIV. Testes free in X and XI. Prostate and accessory glands absent. Spermathecae sessile and diverticula absent, elongated oval or globular between 13/14-17/18, about 0.2-0.3 mm in diameter, two to ten on each side per segment.
Distribution. The new species was found in the river banks of Nan River at Saklek, Phichit, north Thailand, which was covered with worm casts. Etymology. The species was named after the locality on the banks of the Nan River. This was the first time that the worm genus was ever recorded from near this river.
Remarks. For a summary of the following comparisons please see  Description of holotype. Dimensions: body length 131 mm, diameter 2.2 mm in segment VIII, 2.3 mm before the clitellar wing in segment XXIII, 2.4 mm after wing in segment XXX in clitellar region; body cylindrical in anterior part, quadrangular in transverse section behind clitellum. 326 segments. Body color pale brown with variations from red to pink at adjacent tissues of wing portion in different individuals of newly collected specimens. At posterior end dorsal surface considerably broader than the ventral. Clitellar wing on ventro-lateral part of clitellum in XXIV-XXIX, 2.5 mm in height, and 0.5 mm in width on both sides. Prostomium zygolobous.
Septa 5/6-7/8 thicker than between other segments, 8/9-9/10 thick and 10/11 to the last segment thin. Gizzard small, globular in VII. Intestine enlarged from XVI. Hearts, four pairs in VIII-XI. A pair of holonephridia in each segment from segment XIII onwards. Seminal vesicles, four pairs in IX-XII. Ovaries, two pairs in XIII-XIV. Testes free in X and XI. Prostate and accessory glands absent. Spermathecae sessile and diverticula absent, elongated oval or globular between 13/14-15/16, about 0.2-0.3 mm in diameter, four to five on each side per segment.
Distribution. The new species was found at a pond in Satun, south Thailand. Etymology. The name "satunensis" is given in reference to the type locality, Satun.
Distribution. The new species is known from the type locality in the river banks of Mekong River at Wat Hatkai, Chiangkhong, Chiangrai, north Thailand, and was found in several locations along the Mekong River and its tributaries in the northeast and north of Thailand at Buengkan, Loei, Nongkhai, and Chiangrai and in Chaiyaburi, Luangprabang, and Sanakham to Vientiane of Laos.
Distribution. The new species was found in the river banks of Phao River which was covered with worm casts.
Etymology. The species was named after the Phao River, the type locality. Remarks. Glyphidrilus namphao sp. n. is different from G. vangviengensis Chanabun & Panha, 2011 reported from Song River Veintiane, Laos in different locations of wings in XXIV, XXV-XXXI, XXXII, longer clitellum in XIX, XX-XXXV, XXXVI, XXXVII and lacking spermathecae. It differs from G. nanensis sp. n. from Nan River at Saklek, Phichit, north Thailand by G. nanensis sp. n. having wings in XXIV, XXV, XXVI-XXVII, XXVIII, XXIX, a longer clitellum in XVII, XVIII-XXXIII, XXXIV, and spermathecae between 13/14-17/18. It is different from G. sekongensis sp. n. from Ban Kiangkong, Lamarm, Sekong, Laos in the locations of wings in XXV-XXXI, a longer clitellum in XVI, XVII-XXXVI, XXXVII, the genital markings paired or asymmetrical on bc in XVI, XVII-XXIV, XXXII, and spermathecae between 12/13-15/16. Glyphidrilus namphao sp. n. differs from G. chiangraiensis sp. n. from Mekong River at Wat Hatkai, Chiangkhong, Chiangrai, north Thailand in the locations of wings in XXIII, XXIV-XXVI, ½XXVII, XXVII, ½XXVIII, XXVIII, a longer clitellum in XVII, XVIII, XIX, XX, XXI-XXXVI, XXXVII, XXXVIII, XXXIX, and spermathecae between 15/16-20/21 (see Table 1). Diagnosis. Glyphidrilus sekongensis sp. n. is a small sized earthworm with the clitellar wings on the lateral side of the body in XXV-XXXI. Clitellum in XVI, XVII-XXXVI, XXXVII. Female pores, male pores and spermathecal pores not visible. Genital markings: paired or asymmetrical on bc in XVI, XVII-XXIV and XXXII. Four pairs of seminal vesicles in IX-XII. Intestinal origin in XVI. Ovaries in XIII-XIV. Spermathecae between 12/13-15/16. Description of holotype. Dimensions: body length 134 mm, diameter 2.4 mm in segment VIII, 2.3 mm before the clitellar wing in segment XXIV, 2.4 mm after wing in segment XXXII in clitellar region; body cylindrical in anterior part, quadrangular in transverse section behind clitellum. 329 segments. Body color pale brown with variations from red to pink on adjacent tissues of wing portions in different individuals of newly collected specimens. The posterior end on the dorsal surface is considerably broader than the ventral. Clitellar wing on ventro-lateral part of clitellum in XXV-XXXI, 4.0 mm in height, and 0.5 mm in width on both sides. Prostomium zygolobous. Dorsal pores absent. Clitellum annular in XVI-XXXVII. Four pairs of setae per segment from II, setal formula aa:ab:bc:cd:dd = 1.0:0.3:1.0:0.5:1.3 in segment VIII and 1.8:0.8:1.3:0.6:1.5 in postclitellar segments. Female pores, male pores, and spermathecal pores not visible. Genital markings: laterally paired or asymmetrical on bc in XVI-XXIV, XXXII. Septa 5/6-8/9 thicker than between other segments, 9/10-14/15 thick and 15/16 to the last others segment, 9/10-14/15 thick and 15/16 to the last segment thin. Gizzard small, globular in VIII. Intestine enlarged from XVI. Hearts, four pairs in VIII-XI, first in VIII and last in XI. A pair of holonephridia in each segment from segment XIII onwards. Seminal vesicles, four pairs in IX, X, XI, XII. Ovaries, two pairs in XIII and XIV. Testis free in X and XI. Prostate and accessory glands absent. Spermathecae sessile and diverticula absent, small elongated oval or globular between 12/13-15/16, about 0.1-0.3 mm in diameter, two to seven on each side per segment.
Distribution. The new species was found in a stream at Ban Kiangkong, Lamarm, Sekong, Laos. Soils are slightly sandy mixed with black organic matter.
Diagnosis. Glyphidrilus namdonensis sp. n. is small sized earthworm with distinctly expanded tissues, the clitellar wings on the lateral side of the body in XXIV, XXV-XXVIII, XXX. Clitellum in XIX, XX-XXXVI, XXXVII, XXXVIII. Female pores, Septa 5/6-8/9 thicker than between other segments, 9/10-14/15 thick, and 15/16 to the last segment thin. Gizzard small, globular in VIII. Intestine enlarged from XVI. Hearts, five pairs in VII-XI, first in VII and last in XI. A pair of holonephridia in each segment from segment XII onwards. Seminal vesicles, four pairs in IX, X, XI, XII. Ovaries two pairs in XIII and XIV. Testis free in X and XI. Prostate and accessory glands absent. Spermathecae sessile and diverticula absent, small elongated oval or globular between 14/15-18/19, about 0.1-0.2 mm in diameter, two to thirteen on each side per segment.
Distribution. The new species was found only from the banks of Done River at Ban Namdone, Thakhek, Khammouan, Laos.
Septa 4/5-8/9 thicker than between other segments others segment, 9/10-11/12 thick and 12/13 to the last segment thin. Gizzard small, globular in VIII. Intestine enlarged from XVI. Hearts, five pairs in VII-XI, first in VII and last in XI. A pair of holonephridia in each segment from segment XIV onwards. Seminal vesicles, four pairs in IX, X, XI, XII. Ovaries, two pairs in XIII and XIV. Testis free in X and XI. Prostate and accessory glands absent. Spermathecae sessile and diverticula absent, small elongated oval or globular between 14/15-19/20, about 0.1-0.3 mm in diameter, five to thirteen on each side per segment.
Distribution. The new species was found only on the banks of the Mekong River at Ban Khonkhen, Champasak, Laos.
Etymology. The new species was named after Champasak, Laos, the type locality.

Discussion
Semi-aquatic earthworms of the genus Glyphidrilus are widely recorded on the Asian and African continents. Recently, many species have been described from several ecotone areas in Thailand between freshwater and terrestrial habitats of many river basins, where the soil pH varies from neutral to basic at 7-7.5 (Chanabun et al. 2013).
The seven new species from Thailand and Laos presented in this paper range in size, with respect to the other Glyphidrilus members, from large to small, with G. champasakensis sp. n. being the longest and G. nanensis sp. n. the smallest. The other five species are almost of the same size, as shown in Table 1. However, the locations of clitellum, wings, and spermathecae and other characters of the seven species show clear differences from the closely related species.
Glyphidrilus chiangraiensis sp. n. from Mekong River at Wat Hatkai, Chiangkhong, Chiangrai, north Thailand, was found in many areas of the Mekong River and its tributaries from Thailand and Laos, at elevations from 101-385 meters, and co-existing with G. mekongensis Panha & Chanabun, 2012, Amynthas mekongianus (Cognetti, 1922, Amynthas sp., and Metaphire sp. The earthworms and cocoons were found in the soil when digging (Fig. 13) and this is probably the cause of the wide distribution, since cocoons travelled from the high to low elevations during the rainy season. Glyphidrilus nanensis sp. n. was found in the rice fields of a tributary of the Nan River, in north Thailand. It was found co-existing with the common terrestrial earthworm Drawida beddardi (Rosa, 1890). Glyphidrilus nanensis sp. n. is probably an isolated endemic occurring in the Nan River valley. This earthworm was found at soil depths of 5-10 cm in agricultural land. This demonstrates that the species is compatible with at least organic farming and that conservation of the species is not difficult, which is increasingly important in the world. Jouquet et al. (2008b) showed the casts produced by Glyphidrilus sp. can be considered as patches of nutrients in paddy fields. Owa et al. (2003) also observed rice development and greater productivity when earthworm casts were associated to rice plants. Choosai et al. (2010) observed rice development in Northeast Thailand and confirmed the positive effect of earthworms on soil properties, rice yield was higher when presence of casts. This strongly suggests that any agricultural system promoting earthworm development, thereby increasing the number of casts per rice field, could be considered as an useful approach for the sustainable management of paddy fields.
Glyphidrilus satunensis sp. n. was found at a pond, part of a wildlife sanctuary at Satun, south Thailand. It has a similar body size with another species described from nearby in the Malay Peninsula (Chanabun et al. 2013); however, the locations of clitellum, wings, and spermathecae show clear differences between the closely related species.
The four new species described from Laos, G. namphao sp. n., G. sekongensis sp. n., G. namdonensis sp. n., and G. champasakensis sp. n. live in different habitats. Glyphidrilus namphao sp. n. lives in Phao River the river near to the Namphao border between Laos and Vietnam at a higher elevation (525 meters) than the other three new species. Glyphidrilus sekongensis sp. n. lives in a stream located within an evergreen and mossy forest at Ban Kiangkong, Lamarm, Sekong; the forest is covers 70% of the area near the stream. The soils are slightly sandy and black with the accumulation of organic matter. Glyphidrilus namdonensis sp. n. lives in the sandy banks of the Don River, which is one of the branches of Mekong River at Thathek, Khammouan, Laos. Glyphidrilus champasakensis sp. n. is one of the new species along the Mekong River found at a soil depth of 20-40 cm, and has similar characteristics to two other new species (G. sekongensis sp. n. and G. namdonensis sp. n.).
The following key includes only the species most closely related to those described here. The basis for including the species covered in the key is the same as the basis for including previously described species for comparison in the Remarks sections. The remaining species of Glyphidrilus must be identified with other resources. Key to species of the newly described species of Glyphidrilus, and those most similar to them