The semi-aquatic freshwater earthworms of the genus Glyphidrilus Horst, 1889 from Thailand (Oligochaeta, Almidae) with re-descriptions of several species

Abstract The semi-aquatic freshwater earthworm genus Glyphidrilus Horst, 1889 from Thailand was investigated based on extensive recent collecting. The species in this genus were characterized by their external and internal morphological characters of the location of wings, genital openings, genital organ structures and their locations, as well as the dimensions of body length and number of segments. Several type specimens were compared with both previous and newly collected materials. Ten new species are described from several river systems in Thailand; as Glyphidrilus borealis sp. n., Glyphidrilus chaophraya sp. n., Glyphidrilus chiensis sp. n., Glyphidrilus huailuangensis sp. n., Glyphidrilus kratuensis sp. n., Glyphidrilus quadratus sp. n., Glyphidrilus trangensis sp. n., Glyphidrilus wararamensis sp. n., Glyphidrilus vangthongensis sp. n. and Glyphidrilus vesper sp. n. Each species is endemic to a single river system. All 26 previously described species are re-described, and eight lectotypes have been designated. An identification key and a morphological comparison summary are provided.


introduction
Species of the genus Glyphidrilus Horst, 1889 are unfamiliar earthworms living on an ecotone between terrestrial and freshwater ecosystems of rivers, streams, canals, ponds, swamps or even in paddy rice system. Normally the animals orientate vertically head down, with their bodies in the wet soils along the banks, while tails are placed above or on the soil surface. A similar phenomenon has also been reported by Zicsi et al. (1999). The anterior of the body is rounded but the posterior is square (Fig. 1A). In our recent observations the dorsal posterior part of the bodies can be flexed longitudinally as Ushape channels which allow water circulation down the burrow on a large amount of surface area (Fig. 1B). This is probably for oxygen transport to the deeper surface of the worm bodies while worms remain in their burrows, assist the worms to breathe the air directly through the epidermis, such as in the terrestrial worms, but in the semi-aquatic habitats. The function of the peculiar expanded epidermis at about clitellum position called "wings" is still unknown, but Gates (1972) suggested that these vascularized organs evolved for a respiration function in such aquatic habitats. However, because the wings will be deep in the burrow at low oxygen tension, a more conventional explanation might be a use during copulation, like the tubercula pubertatis of some earthworm families. These double functions were also found in the European lumbricid genus Helodrilus (Zicsi et al. 1999). Glyphidrilus exhibits an interesting behavior that is similar to that of some terrestrial earthworm genera such as Drawida and Metaphire. One species of each genus shows seasonal migration depending on wet and dry conditions throughout the year. Glyphidrilus mekongensis Panha and Chanabun, 2012 and Metaphire posthuma (Vaillant, 1868) were observed emerging in 6 November 2010 to 20 January 2012 (Chanabun et al. 2012a) along the Mekong River in northeastern Thailand. Drawida beddardi (Rosa, 1890) species was observed in the same phenomenon in many paddy areas in central Thailand (Chanabun and Panha, personal observations).
After mating Glyphidrilus produces long dark coloured cocoons containing 7-10 juveniles. The cocoons are laid in the wet layer of soils (Fig. 1C).

Taxonomy of Glyphidrilus
Glyphidrilus weberi Horst, 1889, a Javan species, was firstly described. Rosa (1890) described Bilimba papillata from Burma which later was redescribed as G. papillatus (Michaelsen 1896). Glyphidrilus quadrangulus Horst, 1893 from Sumatra and G. kuekenthali Michaelsen, 1896 from Borneo were the next. Later Michaelsen (1897) described G. stuhlmanni from Tanzania, and reported several more Asian species from India, Nepal, Ceylon, Burma, the Andaman Islands, Malay Peninsula, and Thailand (Michaelsen 1902, 1910, Stephenson 1916, 1924, Rao 1922, Gates 1945, 1958, Chen and Xu 1977, Zicsi 1996, Shen and Yeo 2005, Chanabun et al. 2011, 2012a. Because of the unique morphological characteristics of Glyphidrilus, there are some papers about the details of some organs such as Nair (1938) on the anatomy of G. annandalei. Gates (1958) made a detailed description of G. birmanicus from Burma and defined the generic characteristics of the digestive, vascular and excretory systems. Jamieson (1968) added G. ugandaensis from Uganda which has a distinctively high number of spermathecae and small longitudinal lamellar ridges (wings) -more like a long tubercula pubertatis. Later Brinkhurst and Jamieson (1971) re-classified it as Callidrilus. Gates (1972) tried to redefine the characteristics of Glyphidrilus and stated that "The glyphidrile wings are richly vascularized which suggests a respiratory function for those organs which are, however, developed only just before sexual maturity" Respiration is necessary for all life stages of the worms. Therefore we do not accept Gates' interpretation of the function of the wings.
There are 25 species and one subspecies of Glyphidrilus recorded (Zicsi 1996, Chen and Xu 1977, Shen and Yeo 2005, Chanabun et al. 2011, 2012a, all from Asia and Africa. The records in Asia are numerous from the Indonesian islands through the Malay Peninsula and Burma, west to India and Nepal, and north to China. The near-est records to Thailand are five new species from Singapore and Malaysia (Shen andYeo 2005, Chanabun et al. 2012a, b), one species described from China (Chen and Xu 1977), and a species described from Laos (Chanabun et al. 2011). Here, descriptions of ten new species found in various parts of Thailand, with habitat characteristics of each species including grain size of soil particles contained in habitats of some described species. The redescriptions and illustrations of some previously described species will also be presented and discussed.

Thailand river systems and habitat characteristics of Glyphidrilus
The habitat that is vital to the semi-aquatic earthworms and other aquatic animals is water. Within Thailand water has an uneven and inequitable spatial and temporal distribution, the latter related to the strongly monsoonal climate regime. The spatial distribution of rivers and their headwaters has not been static over long periods of time, but rather has changed. There are two major river systems in Thailand, the Chao Phraya and the Mekong Rivers that drain to the Gulf of Thailand and the South China Sea, respectively. In addition, Thailand has smaller river systems that drain to the Gulf of Thailand and the Andaman Sea (Fig. 2). The Mekong River is unlikely to have assumed its present course along the eastern border of the Khorat Plateau until the late Pleistocene epoch (Hutchinson 1989). Other changes in flow patterns are depicted in Figure 2. Like other geomorphological changes, reorganization of rivers can cause allopatric speciation of aquatic or semi-aquatic fauna. As such, the water systems are probably related to Glyphidrilus speciation and distribution, whilst the morphological characteristics of animal bodies and reproductive characters, such as the mixed round-square body shape and also the spindle shaped cocoon buried in the soil, probably inhibit the dispersal of the animals. Glyphidrilus biodiversity has probably been influenced by river system changes, given that these worms live in or near waterfalls, small streams, rivers, canals, swamps, and paddy systems (Figs 3,4). The interconnections among these diverse microhabitats allow gene flow, but when connections are severed, independent evolution in isolation can lead to speciation. As discussed later, there are some endemic species within each drainage system, but there are also some wider distributed species.

Materials and methods
Earthworms were sampled from several localities in Thailand by carefully digging up the topsoil near casts on the shore and in the water using hand sorting and sieving the soil from banks of freshwater habitats. The worms were killed in 30% (v/v) ethanol, photographed, transferred to 5% (w/v) formalin for fixation for approximately 12 hours, and then transferred to 70% (v/v) ethanol for longer term preservation and subsequent morphological studies. Some specimens were placed in 95% ethanol for further DNA analysis. Major course changes along the Mekong River during the late Cenozoic era. Prehistoric river courses are shown in blue lines, and the name of the captured river are shown along with the approximated time (in million years ago) in parentheses. Based on the map of Hutchinson (1989), the current rivers are shown in dark lines with their names.
Specimens were examined and compared with reference materials from several natural history museums.
The descriptions were made from observations under an OLYMPUS SZX16 stereoscopic microscope. The following external and internal morphological characters were recorded: body length and segment number, the positions of clitellum and wings, genital markings, intestinal origin, gizzard, spermathecae, hearts and seminal vesicles. Drawings were made of the body segments and the distinctive external characters and internal organs. The number of segments and the body width and length were measured in both full adults and juveniles, and are presented as the ranges (min-max) and mean±one standard deviation (SD).

Some important terminology for the morphological characters of Glyphidrilus
The terms normally use for descriptions of earthworm characters were compiled and modified from the literature (Gates 1972, Edwards and Bohlen 1996, Sims and Gerard 1999, are arranged in alphabetical order. Some are also shown in Figure 5. Annular: Clitellum encircling the body, being continuous ventrally and equally developed. Clitellum: The region of the body wall formed from several layers of highly glandular epidermal cells, used for secreting mucus from which the egg capsule is formed. It provides nourishment for the developing embryos and, in some species, is used to hold partners together during copulation. Dorsal pore: An intersegmental mid-dorsal pore, the sphinctered aperture leading from the coelom to the exterior between some segments. Genital markings (gm): The area of modified epidermis important during copulation. Gizzard (gi): Part of the alimentary canal having thick muscular walls, its main function is to grind up food before it reaches the intestine. Hearts (he) or pseudohearts or lateral commissures or lateral hearts: Paired segmental blood vessels conveying the blood lateral-ventrally from the dorsal vessel to the ventral or sub-neural vessel.

Nephridia (np):
Excretory organ, paired in most segments and comprised of a ciliated funnel or nephrosome opening into the preceding segment and leading into a system of tubules that are richly supplied with blood vessels, and terminating in a vesicle or bladder before discharging to the exterior through a nephridiopore in the body wall. Peristomium: The foremost true segment of an earthworm which bears the mouth. Prostate: A paired gland in terrestrial earthworms that produces a fluid for transporting and nourishing sperm during copulation; either associated with the vas defer- ens or opening with a separated duct discharging through or nearby a male pore. This organ never occurrs in Glyphidrilus. Prostomium: The portion of the head in earthworms that is situated in front of the mouth. The pre-segmental lobe is developed into a proboscis that is used for probing and swallowing soils and leaf litter. It is believed to be highly chemosensory. Seminal vesicle(s) (sv): Pouch formed in a septum adjacent to a testicular segment, where the spermatogonia undergo the later stages of spermatogenesis and are stored until required during copulation. Setae: Bristle of chitin, the product of a single ectodermal cell, used for locomotion, for gripping the walls of a burrow or for holding another individual during copulation. Spermatheca or spermathecae (sc): Flask-like invagination of the body wall for the reception and storage of sperm from a partner during copulation.

Anatomical abbreviations
The following abbreviations used in the descriptions of anatomy are as appeared in Bantaowong et al. (2011a, b), Chanabun et al. (2011Chanabun et al. ( , 2012a Diagnosis. Prostomium zygolobous. Body shape nearly circular in cross section in anterior part, and becoming quadrangular in posterior part or behind clitellum. Anus dorsal or dorso-terminal. A longitudinal lamellar ridge at maturity from body wall on each side, in bc, through several of the clitellar segments (wi). Dorsal pores absent. Se-tae four pairs per segment. Clitellum annular. Genital apertures, all minute and superficial. Male pores inconspicuous, located ventral to wi. Spermathecal pores usually all behind the testis segments. Gi in 7 or 8 sometime extending into an adjacent segment; strengthening of the musculature at the beginning of the intestine present or absent, no well marked intestinal gi. Calciferous glands absent. Sv usually short, each confined to segment of origin or one more; usually four pairs in 9-12. Holonephridia. Nephrostomes avesiculate and without sphincters or intestinal caeca. Testis and funnels free in 10 and 11; male ducts intramural. Ov fan shaped and with several egg strings. Ovisacs present or absent. Prostate glands absent. Sc adiverticulate.
Distribution. The genus Glyphidrilus has widely distributed range from Tanzania in Africa to South Asia and Southeast Asia. In Asia many species were recorded from Yunnan, southwest China, Hainan, Indochina, Malaysia, Laos, Thailand, Singapore, and some Sunda Shelf islands such as Sumatra, Java, Borneo and Celebes (Horst 1893, Chen 1938, Gates 1958, 1972, Chen and Xu 1977, Shen and Yeo 2005, Chanabun et al. 2011, 2012a. It occurs along the river banks, lakes, canals, swamps and in paddy rice systems. Material examined. The specimens which closely matched to the measurements and anatomical characters with the original description is designated herein as the lectotype ZMH V5097 (Fig. 6). The type locality of this species is Buitenzorg Java. Paralecto type: ZMH V5097.1 (2 adults) same locality with lectotype. Lectotype designation proposed herewith is necessary to ensure the species is based on a complete adult earthworm.
Variation. Additional specimens (5 complete  Material examined. The specimen with the measurements and anatomical characters agreeing with the original description is designated as the lectotype ZMH V5875 (Fig.  8). The type locality of this species is Malay Peninsula, Lubock Paku, Pahang River. Paralectotype: ZMH V5875.1 (only one tail) same locality with lectotype. Lectotype designation proposed herewith is necessary to ensure the species is based on a complete adult earthworm.

Glyphidrilus spelaeotes
Remarks. See Table 1. This species is known only from Siju Cave, Garo Hills, Assam as in the original description. In the recent collections there are no records of this species.  Stephenson, 1930 http://species-id.net/wiki/Glyphidrilus_horsti Fig.15, Table 1 Glyphidrilus horsti Stephenson, 1930: 4    Etymology. The specific epithet "borealis" refers to the occurrence of the new species principally in the northern mountain ranges of Thailand, in various watersheds of four river basins.
Remarks. Glyphidrilus borealis sp. n. is similar to G. birmanicus from Burma in the locations of wi, however G. birmanicus has a longer clitellum in 12, 13-43, 44, np from 13, and sc in 13/14-17/18. It is similar to G. jacobsoni from Sumatra in the locations of wi, but G. jacobsoni has a bit shorter clitellum in 18-31; four pairs of he in 8-11, and sc in 12/13-16/17.
Description of Holotype. Dimensions: body length 116 mm, diameter 7.5 mm in segment 8 and 8.0 mm before the clitellar wi in segment 24, 8.2 mm after wi in segment 32 in clitellar region; body cylindrical in anterior part, quadrangular in transverse section behind clitellum. 251 segments. Body colour pale brown with variations from red to pink at adjacent tissues of wi portion in different individuals of newly collected specimens. At posterior end dorsal surface considerably broader than the ventral. Clitellar wi on ventro-lateral part of clitellum in 25-31, 3.1 mm in height, and about 0.4 mm in width on both sides. Prostomium zygolobous. Dorsal pores absent. Clitellum annular in 12-40. Four pairs of setae per segment from 2, setal formula aa:ab:bc:cd:dd = 2.0:0.5:1.8:2.0:2.8 in segment 8 and 1.5:0.4:1.6:0.6:2.1 in postclitellar segments. Female pores, male pores and spermathecal pores not visible. Gm laterally paired or asymmetrical on bc in 15-17, 19, 24, and 32, and median paired on aa in 12-13 and 32-33.
Variation. Body lengths of adult paratypes (16)  Distribution. The new species is known from the type locality in the river banks of Sakulnothayan waterfall and Wanathara Resort, Vangthong, and Chadthakan waterfall, Chadthakan, Phitsanulok, Thailand. This species was found on the shore but in proximity to the river water, in loamy sand topsoil (88.2% sand, 10.2% silt, 1.6% clay, pH 7.5-7.6) and also under the water at about 10-15 cm depth. The soil surface was covered with worm casts near the river banks.

Glyphidrilus chaophraya
Etymology. This new species was named after the Chaophraya River, which is a famous and important river in central Thailand. This is the first time Glyphidrilus has been recorded from this river.
Description of Holotype. Dimensions: body length 116 mm, diameter 2.7 mm in segment 8, 4.4 mm before the clitellar wi in segment 23, 3.9 mm after wi in segment 33 in clitellar region; body cylindrical in anterior part, quadrangular in transverse section behind clitellum. 325 segments. Body colour pale brown with variations from red to pink at adjacent tissues of wi portion in different individuals of newly collected specimens. At posterior end dorsal surface considerably broader than the ventral. Cli-tellar wi on ventro-lateral part of clitellum in 24-32, 4.6 mm in height, and about 0.6 mm in width on both sides. Prostomium zygolobous. Dorsal pores absent. Clitellum annular in 20-43. Four pairs of setae per segment from 2, setal formula aa:ab:bc:cd:dd = 1.6:0.6:1.2:0.6:2.4 in segment 8 and 1.6:0.5:1.2:0.5:2.1 in postclitellar segments. Female pores, male pores and spermathecal pores not visible. Gm: laterally paired or asymmetrical on bc in 20-23 and median paired on aa in 13, 14 and 34.
Septa 5/6-7/8 thicker, 8/9-10/11 thick and 11/12 to the last segment thin. Small globular gi within 8. Intestine enlarged from 15. Dorsal blood vessel anterior to 7. He in 7-11. No distinguishable np in first fourteen segments. Sv in 9-12, pair in segment 12 larger than the others. Ov in 13-14. Prostate and accessory glands absent. Sc in 16/17-22/23, about 0.1-0.3 mm in diameter, one to thirteen on each side per segment. Distribution. The new species is known from the type locality in the river banks of Chaophraya River, Payuhakiri, Nakhonsawan, Thailand. This species was found on the shore but in proximity to the river water, in the loamy sand topsoil (88.2% sand, 10.2% silt, 1.6% clay, pH 7.5-7.6) and also under the water at about 20-30 cm depth. The soil surface was covered with worm casts near the river banks.
Description of Holotype. Dimensions: body length 71 mm, diameter 10.0 mm in segment 8, 11.0 mm before clitellar wi in segment 23, 11.0 mm after wi in segment 30 within the clitellum; body cylindrical in anterior part, quadrangular in transverse section behind clitellum. 274 segments. Body colour pale brown with variations from red to pink at adjacent tissues of wi portion in different individuals of newly collected specimens after placement in 30% ethanol for narcotization. Dorsal surface considerably broader than the ventral at posterior end. Clitellar wi on ventro-lateral part of clitellum in 24-29, 3.2 mm in height, and about 0.3 mm in width on both sides. Prostomium zygolobous. Dorsal pores absent. Clitellum annular in 15-33. Four pairs of setae per segment from 2, setal formula aa:ab:bc:cd:dd = 1.1:0.5:1.1:0.4:1.8 in segment 8 and 1.2:0.4:1.1:0.6:2.0 in postclitellar segments. Female pores, male pores and spermathecal pores not visible. Gm: median paired on aa in 13-14, lateral paired on bc in 15-23.

Glyphidrilus huailuangensis
Etymology. The name "huailuangensis" is given to this species for its type locality in the Huailung waterfall, Ubon Ratchathani. This is the first record of this genus at this locality.
Description of Holotype. Dimensions: body length 91 mm, diameter 2.5 mm in segment 8, 1.9 mm before the clitellar wi in segment 25, 1.8 mm behind wi in segment 31 within the clitellar region. Body cylindrical in anterior part, quadrangular in transverse section behind clitellum. 228 segments. Body colour pale brown with variations from red to pink at adjacent tissues of wi portion in different individuals of newly collected specimens after placement in 30% ethanol for narcotization. Dorsal surface considerably broader than the ventral at posterior end. Clitellar wi on ventro-lateral part of clitellum in 26-30, 2.3 mm in height, and about 2.9 mm in width on both sides. Prostomium zygolobous. Dorsal pores absent. Clitellum annular in 12-32. Four pairs of setae per segment from 2, setal formula aa:ab:bc:cd:dd = 1.0:0.5:1.5:0.5:1.0 at 8 and 1.2:0.5:1.6:0.5:1.5 in postclitellar segments. Female pores, male pores, and spermathecal pores not visible. Gm: lateral paired or asymmetrical on bc in 16, 17-24, median paired on aa in 31.
Distribution. The new species is known only from the type locality. This species was found on the shore near water in sandy loam topsoil (80% sand, 13.1% silt, 5.6% clay, pH 7.5) and in the water at about 5-10 cm depth. The soil surface was covered with worm casts.
Etymology. The name "wararamensis" is given to this species for its type locality at the stream near Wattham Wararam, Phanom, Suratthani, Thailand. The locality is a part of this famous stream and is the first record of this genus at this locality.

Glyphidrilus trangensis
Etymology. The name "trangensis" is given to this species as it is the principal name of the type locality of the new species.
Description of Holotype. Dimensions: body length 32.5 + mm, diameter 2.2 mm in segment 8, 2.2 mm before clitellar wi in segment 20, 2.2 mm behind wi in segment 28 in intraclitellar region. Body cylindrical in anterior part, quadrangular in transverse section behind clitellum. 173 + segments. Body colour pale brown with variations from red to pink at adjacent tissues of wi portion in different individuals of newly collected specimens after placement in 30% ethanol for narcotization. Dorsal surface considerably broader than the ventral at posterior end. Clitellar wi on ventro-lateral part of clitellum in 23-27, 2.2 mm in height, and about 0.6 mm in width on both sides. Septa 5/6-8/9 thicker, 9/10-14/15 thick and 15/16 to the last segment thin. Gi small globular within 8-9. Intestine enlarged from 16. Dorsal blood vessel aborted anterior to 8. He in 8-11. No distinguishable np in first eleven segments. Sv in 11-13.
Distribution. The new species is known only from the type locality in a muddy swamp near Trang River, Nayong, Trang, Thailand. Collections in nearby areas have found other Glyphidrilus species but not this species. This new species was found on the shore connected to water of the sandy mud topsoil covered with worm casts and in the water at about 5-8 cm depth. Remarks. Glyphidrilus trangensis sp. n. similar to G. horsti from Pulau Berhala, Straits of Malacca in the locations of wi, whereas G. horsti has clitellum in 17, ½18, 18, 19-29, 30, ½31, he in 8, 9-11, and sc in 14-17 or 14/15-17/18. The new species is similar to G. birmanicus from Burma in the locations of wi however, G. birmanicus has longer clitellum in 12, 13-43, 44, he in 7-11, and sc in 13/14-17/18.

Glyphidrilus kratuensis
Etymology. The name "kratuensis" is given to this species for its type locality in the Kratu waterfall, Kratu, Phuket, Thailand. The locality is a part of this famous waterfall and the first record of this genus in this location.
Description of Holotype. Dimensions: body length 83 mm, diameter 2.4 mm in segment 8, 2.9 mm before clitellar wi in segment 22, 2.8 mm behind wi in segment 30 in intraclitellar region. Body cylindrical in anterior part, quadrangular in transverse section behind clitellum. 235 segments. Body colour pale brown with variations from red to pink at adjacent tissues of wi portion in different individuals of newly collected specimens after placement in 30% ethanol for narcotization. Dorsal surface considerably broader than the ventral at posterior end. Clitellar wi on ventro-lateral part of clitellum in 23-28, 29, 2.4 mm in height, and about 0.6 mm in width on both sides. Prostomium zygolobous. Dorsal pores absent. Clitellum annular in 18-31. Four pairs of setae per segment start from 2, setal formula aa:ab:bc:cd:dd=1.0:0.6:1.7:0.7:1.1 in segment 8 and 0.9:0.4:0.8:0.4:1.0 in postclitellar segments. Female pores, male pores, and spermathecal pores are not visible. Gm: lateral paired or asymmetrical on bc in 15-16, 29, median paired on aa in 19-22 and 30-31.

Discussion
The semi-aquatic freshwater earthworm genus Glyphidrilus is recorded only in Africa and Asia. In Thailand, it occurs in various types of natural freshwater habitats of rivers, streams, canals, ponds and even in paddy areas. The worms normally live in the topsoil near the edge of water systems adjacent to terrestrial habitats in soil types that vary from muddy to sandy sediments, and with a pH ranging from neutral to very mild basic conditions. The earthworms treated herein were all found at a depth of 5-15 cm from the surface in wet top soil and usually near casts. The worms excrete casts on the soil surface by locating the tails up to the soil surface while the heads go down to deeper soils.
The species identifications in this paper followed the concepts from the principal papers presented by Horst (1889), Michaelsen (1902), Brinkhurst and Jamieson (1971), Gates (1972), Shen and Yeo (2005) and the very recent publications by Chanabun et al. (2011Chanabun et al. ( , 2012a. The external morphological characters of the locations of wings and clitellum, the arrangement and position of genital markings, and internal characters, such as digestive, reproductive and circulatory organs, are very significant (informative and diagnostic) for species identification. However the wings and clitellar position are highly variable in all aquatic groups (Alma, Sparganophilus, Criodrilus) (Brinkhurst and Jamieson 1971), and most probably shows a seasonal pattern. The ten new species of Glyphidrilus described herein were all found in different areas in Thailand, and the habitat types are very diverse. Although all the new species look superficially similar to the previously described Southeast Asian species, there are distinct variations in both their external and internal morphological characteristics.
The ten new species ranged in size, with respect to other Glyphidrilus members, from large to small, of which G. vangthongensis sp. n. from Phitsanulok from the north was the longest, G. borealis sp. n. from Chiangmai, G. chaophraya sp. n. from Nakhonsawan and the three species from the Northeast (G. chiensis sp. n. from Mahasarakham, G. quadratus sp. n. and G. huailuangensis sp. n. from Ubon Ratchathani) were of medium to large size, but larger than the three species from the south (G. trangensis sp. n., G. kratuensis sp. n., and G. wararamensis sp. n.) and the single species from the west (G. vesper sp. n.) of Thailand.
The history and geography of the river systems are probably important to Glyphidrilus speciation. All the ten proposed new species described here appear to be highly endemic with very little range overlap between them, and most are confined to a small region (Figs 37,38). Glyphidrilus borealis sp. n., found in the northern most watershed of the Ping River at about 400-700 m amsl was also found in a hot spring area in Chiang Rai province but was the only species found in an almost 100 x 100 km section of northern Thailand (Fig. 37). Glyphidrilus vesper sp. n. occupies the western area along the Myanmar border with one locality shared with G. borealis sp. n. in the Ping River but only in the lowest part of the river at the southern margin of the G. borealis sp. n. range. Its range covers almost 400 km from north to south, starting from the Maeklong watershed to the Maeklong River and tributaries. Glyphidrilus chiensis sp. n. occupies the northeast part of the Chi River system, which drains into the Mekong River, covering all the huge upper northeastern area at altitudes of about 150-250 km (Fig. 37). This species was mostly collected within paddy systems. To the south of that range G. quadratus sp. n. was found in the southern half of Northeast Thailand along the Mun watershed, Mun River and its tributaries, as well as in the eastern region in the Chantaburi watershed and river systems along the eastern Gulf of Thailand. It appears to be the only species of this genus in that area. Glyphidrilus chiensis sp. n. has a range spanning almost 900 km ( Fig. 37). Glyphidrilus vangthongensis sp. n. occupies the central lowlands to almost the upper gulf drainage, spanning almost 600 km (Fig. 37). Glyphidrilus wararamensis sp. n. is highly endemic, occurring only in a small area of the southern peninsula on the Tapi watershed (Fig. 38). These mainly allopatric distributions suggest that geographic speciation could be important and potentially explain the evolution of this genus in SE Asia. However, molecular analysis will be an effective tool for revealing the evolutionary story of Glyphidrilus, in combination with taxonomic and geographic information. Additional data from the other species, including individuals from multiple sites across their apparent ranges (for intra-species variation) for each species, are needed before we can support their morphological species delimitations and test hypotheses about geographic variation, geographic speciation, and the effects of river drainage history. Accordingly, additional sampling in many new sample sites is now being undertaken.