Morphological and molecular evidence reveal a new species of the earthworm genus Pontodrilus Perrier, 1874 (Clitellata, Megascolecidae) from Thailand and Peninsular Malaysia Author Seesamut, Teerapong Author Sutcharit, Chirasak Author Jirapatrasilp, Parin Author Chanabun, Ratmanee Author Panha, Somsak text Zootaxa 2018 2018-10-05 4496 1 218 237 journal article 29266 10.11646/zootaxa.4496.1.18 de8bc64e-676c-49e3-a3b1-8dd1beb72604 1175-5326 1446765 44FD2217-E2B2-4F73-B813-96FB78E46771 Pontodrilus longissimus Seesamut & Panha , sp. n. ( Figures 4 , 5 , 7A ; Table 3) Type specimens. Holotype : CUMZ 3670 ( Fig. 4 ), Hat Pak Meng , Sikao , Trang , Thailand ( 7° 30' 14.3" N , 99° 19' 06.8" E , 7 m above mean sea level), coll. T. Seesamut , C. Sutcharit , R. Srisonchai & A. Pholyotha , 30 Aug 2015 . Paratypes : CUMZ 3671 , 64 adults and 37 juveniles ; NHMUK , 2 adults ; ZMH , 2 adults ; same collection data as holotype . Other materials examined. Thailand . 23 adults and 57 juveniles , CUMZ 3657 , Hat Chao Lao , Thamai , Chanthaburi ( 12° 33' 52.8" N , 101° 54' 25.1" E ) on 8 August 2015 . 4 juveniles , CUMZ 3658 , Ban Koh Kaew Naruemit , Pak Phanang , Nakhon Si Thammarat ( 8° 14' 28.1" N , 100° 16' 41.2" E ) on 3 September 2015 . 17 juveniles , CUMZ 3659 , Klong Bang Siap , Patiew , Chumphon ( 10° 39' 29.9" N , 99° 18' 39.1" E ) on 3 September 2015 . 33 adults and 67 juveniles , CUMZ 3660 , Hat Mai Khao , Talang , Phuket ( 8° 05' 47.9" N , 98° 17' 55.5" E ) on 5 December 2015 . 30 adults and 40 juveniles , CUMZ 3661 , Nang Thong Bay Resort, Takua Pa, Phangnga ( 8° 38' 43.4" N , 98° 14' 50.1" E ) on 5 December 2015 . 3 adults and 12 juveniles , CUMZ 3662 , Hat Bang Sak , Takua Pa , Phangnga ( 8° 47' 03.4" N , 98° 15' 46.1" E ) on 5 December 2015 . 5 adults and 11 juveniles , CUMZ 3663 , Hat Sai Ngoen , Klong Yai , Trat ( 12° 02' 43.5" N , 102° 45' 01.8" E ) on 15 December 2015 . 6 adults and 33 juveniles , CUMZ 3664 , Hat Koey , Kaper , Ranong ( 9° 37' 26.7" N , 98° 28' 08.6" E ) on 10 January 2016 . 74 juveniles , CUMZ 3665 , Hat Yao , Nuea Klong , Krabi ( 7° 58' 49.4" N , 98° 56' 46.6" E ) on 11 January 2016 . 20 juveniles , CUMZ 3666 , Hat Samran , Hat Samran , Trang ( 7° 14' 02.9" N , 99° 32' 19.4" E ) on 12 January 2016 . 29 adults and 45 juveniles , CUMZ 3667 , Hat Bo Chet Look , La Ngu , Satun ( 6° 53' 32.6" N , 99° 41' 12.5" E ) on 12 January 2016 . Malaysia . 10 juveniles , CUMZ 3668 , Pantai Chendor , Cukai , Terengganu ( 4° 10' 36.7" N , 103° 25' 15.6" E ) on 25 January 2016 . 1 juvenile , CUMZ 3669 , Pantai Melawi , Sungai Ger Bachok , Kelantan ( 5° 59' 35.7" N , 102° 25' 54.2" E ) on 26 January 2016 . Description of holotype . Length 161 mm , diameter 3.47 mm at segment X, 3.10 at segment XX, 3.90 mm at clitellum, body cylindrical with 183 segments. The body colour ranges from red to pink and pale brown around clitellum in newly collected specimens after placement in 30% (v/v) ethanol for narcotization. Prostomium epilobous. Clitellum saddle in XIII–XVII, setae present. Setae lumbricine, ab absent on XVIII, aa:ab:bc:cd:dd = 1.8:0.3:1.6:1.1:5.0 in XX. Female pores paired, medio-ventral in XIV. Male pores paired in XVIII, distance between male pores 2 mm , penial setae absent (or not found). Spermathecal pores intersegmental two pairs in 7/8 and 8/9, ventrolateral, in line with setae b, distance between spermathecal pores 2 mm . Dorsal pores absent. Genital markings present at intersegmental boundary in 17/18 and 18/19. Septa 4/5–12/13 thickened. Gizzard and calciferous glands absent or not developed. Intestine enlarged from XV. Intestinal typhlosole absent. Esophageal hearts eight pairs in VI–XIII. No nephridia distinguishable in first fourteen segments, beginning in XV with a pair of coiled tubules. Two pairs of spermathecae in VIII and IX. Ampulla as large ovoid sac, without diverticulum. Two pairs of seminal vesicles in XI and XII. No mature (iridescent) sperm observed in spermathecae or seminal vesicles. Ovary in XIII. Tubular prostate single pair, muscular duct in XVIII. Accessory glands absent. Variation. Holotype measures 161 mm body length with 183 segments. Body length of paratypes and nontypes (adult specimens) range in size from 125–165 mm , with 160–191 segments. Clitellum in XIII, XIV–XVII. Intestine origin in XV–XVII. Nephridia from XIII or XV. Distribution. The new species is known from the type locality and along the coastal areas of Thailand , both the Gulf of Thailand and the Andaman Sea, and also some parts of Peninsular Malaysia ( Fig. 1 ). Habitat. Found in the coastal salt marsh of estuaries at about 10–30 cm depth, in mud with a high content of organic matter and a salinity ranges from 1–33 ppt. Etymology. The specific epithet “ longissimus ” in Latin means “the longest”. This name refers to the length of this species compared to the other species in this genus. Diagnosis. Length 125–165 mm , 160–191 segments. Setae lumbricine, penial setae absent. Prostomium epilobous. Dorsal pores totally absent. Clitellum saddle shape, in XIII, XIV–XVII. Male pores paired in XVIII; female pores paired in XIV. Genital markings present in 17/18 and 18/19. Spermathecae as large ovoid sacs in VIII and IX, without diverticulum. Two pairs of seminal vesicles in XI and XII. Ovary in XIII. Esophageal hearts eight pairs in VI–XIII. Tubular prostates in XVIII. TABLE 2. Comparison of Pontodrilus litoralis from Thailanđ anđ Peninsular Malaysia with nominal species of Pontodrilus đescribeđ from that area anđ now in synonymy with P. litoralis . Character P. litoralis Cryptodrilus insularis P. ephippiger P. matsushimensis P. laccadivensis P. albanyensis ( Grube, 1855 ) * Rosa, 1891 Rosa, 1898 Iizuka,1898 Beđđarđ, 1903 Michaelsen, 1907a ? Characters not given in the original đescription; * Specimen from Thailanđ anđ Peninsular Malaysia ;

Length (mm)	28.136	50	43.47	90.110	90.110	50
Segment	76.128	100	85.100	100.105	?	86
Clitellum	XIII.XVII, XVIII	?	XIII.XVII	XII.XVII	XIII.XVII	?
Spermathecal pore	7/8.8/9	7/8.8/9	7/8.8/9	7/8.8/9	7/8.8/9	7/8.8/9
Genital marking	19/20 or 20/21	?	XIX.XX	XIX.XX	XII/XIII, XIV; XI/XII, XII/XIII	17/18, 18/19
First nephriđia	XIII	XIII	?	XIII	XIII	?
Last heart	XIII	?	XIII	XIII	XIII	?
Intestinal origin	XVI or XVII	?	XVIII	XIV	?	?
Spermathecae	VIII.IX	VIII.IX	VIII.IX	VIII.IX	?**	?**
Diverticulum	slenđer	absent	narrow tubular	tubular	tubular	tubular
Type locality	Villa Franca on the French Riviera, France	Aru Islanđ, Inđonesia	Christmas Islanđ, Australia	Matsushima Bay, Rikuzen, Japan	Laccađive anđ Malđive Islanđs	Princess Royal Harbor, Australia
? Characters not given in the original đescription; * Specimens from Thailanđ anđ Peninsular Malaysia; ** Spermathecal pores at 7/8, 8/9.
TABLE ³. Comparison of the morphological characteristics of P. longissimus sp. n. with other species of the genus.
Character	P. litoralis * (Grube, 1855)	P. lacustris (Benham, 1903)	P. agnesae Stephenson, 1915	P. sinensis Chen & Zhifang, 1977	P. primoris Blakemore, 2000	P. longissimus sp. n. *
Length (mm)	28.136	35.40	65	30.5.41	60.120 (120)	125.165
Segments	76.128	85.90	116	64.94	100.150 (118)	160.191
Clitellum	XIII.XVII, XVIII	½XIII.XIV	½XIII.½XVII	XIII.XVIII	absent	XIII, XIV.XVII
Penial setae	absent	present	present	?	absent	absent
Spermathecal pore	7/8.8/9	VI.IX	7/8.8/9	7/8.8/9	7/8.8/9	7/8.8/9
Genital marking	19/20 or 20/21	?	absent	?	XIV.XVIII, 18/19. 19/20	17/18.18/19
First nephriđia	XIII	XV	XII or XIII	XIII	XIV	XIII or XV
Seminal vesicle	XI anđ XII	IX anđ XI	IX anđ XII	IX.XII	XI anđ XII	XI anđ XII
Intestinal origin	XVI or XVII	XIII	?	XIV or XV	XV	XV or XVI or XVII
Spermathecae Diverticulum	VIII.IX slenđer	VI.IX globular	? club-shapeđ	??	VIII.IX short	VIII.IX absent
Type locality	Villa Franca, French Riviera, France	Lake Wakatipu, South Islanđ, New Zealanđ	Sri Lanka	Kunming, Yunnan, China	Tasmania, Australia Hat Pak Meng, Sikao, Trang, Thailanđ

FIGURE 4. External and internal morphology of the holotype (CUMZ 3670) of Pontodrilus longissimus sp. n. ; A. External ventral view; B. Internal dorsal view; C. Spermatheca. FIGURE 5. Photographs showing: A. Pontodrilus longissimus sp. n. (CUMZ 3670, CUMZ 3671) just after the first step preservation in 30% (v/v) ethanol; B. Ventral view of anterior portion; C. Cocoons; D. The type locality of Pontodrilus longissimus sp. n. at Hat Pak Meng, Sikao, Trang, Thailand. FIGURE 6 . Bayesian inference tree based on the partial COI sequence (658 bp) of two Pontodrilus species and outgroups. Bootstrap values of>70% and posterior probability values of>0.95 are shown on the tree as ML/BI values. Specimen names correspond to those in Table 1. Scale bar represents the number of nucleotide substitutions per site. Remarks. The new species, P. longissimus sp. n. , differs from the cosmopolitan littoral species P. litoralis , based on the specimens from Thailand and Peninsular Malaysia , by differences in segment number (160–191 and 76–128 for P. longissimus sp. n. and P. litoralis , respectively), and body length ( 125–165 mm and 28–136 mm for P. longissimus sp. n. and P. litoralis , respectively). Additionally, they are easily distinguished by the spermathecal diverticulum, absent in the new species but present in all other currently accepted species of the genus. Pontodrilus litoralis has a long and slender diverticulum, P. lacustris has a small globular diverticulum, P. agnesae has a spindle or club-shaped diverticulum, P. primoris has a short diverticulum relative to the ampulla. Among the many descriptions of P. litoralis and its synonyms, only P. insularis ( Rosa, 1891 ) from Aru Islands, Indonesia (“ Insel Aru”) is without spermathecal diverticula. Michaelsen (1897) identified tentatively two specimens from Sri Lanka (“ Ceylon ”) as P. insularis but conjectured that the absence of diverticula may be due to the fact that his and Rosa's specimens were subadult without clitellum, and he suggested synonymy with P. bermudensis Beddard, 1891 , considering their overall similarity. Beddard (1895) also noted that the specimens were immature. However, Rosa (1898) maintained the possibility that the absence of diverticula is a taxonomic character and not caused by the immature state of the specimens, since “even in a series of sections no traces could be found of an organ which in the adult reaches so great a development” (id.: 283). Nevertheless, Michaelsen (1910) established the synonymy with P. bermudensis . Later, Easton (1984) synonymized P. bermudensis with P. litoralis . Even though Michaelsen's decision was not questioned in the subsequent taxonomic literature, the possibility remains that Rosa's species is distinct from P. litoralis (see Rosa 1898 ) and that it is characterized by the absence of diverticula. Pontodrilus insularis as originally described is much smaller than P. longissimus sp. n. (length 50 mm , diameter 3 mm , ca. 100 segments) and the first nephridia are found in XIII. Pontodrilus longissimus sp. n. is easily distinguished from other Pontodrilus species by the morphometric characteristics shown in Table 3. Cocoons, presumably of this earthworm, were collected from the littoral zone of the Andaman Sea at Hat Bo Chet Look, La Ngu, Satun . The cocoons are lemon-shaped, yellowish green in colour ( Fig. 5C ).