Pheretima vungtauensis, Nguyen & Nguyen & Nguyen, 2018

Nguyen, Tung T., Nguyen, Nam Q. & Nguyen, Anh D., 2018, First record of the earthworm genus Pheretima Kinberg, 1867 sensu stricto in Vietnam, with description of a new species (Annelida: Clitellata: Megascolecidae), Zootaxa 4496 (1), pp. 251-258 : 252-257

Pheretima vungtauensis sp. nov.

( Figure 1 )

Material examined. HOLOTYPE: mature Specimen (CTU-EW.166.h01) banana plantation (10o44’57.6 N; 107o14’35.1 E), 206 m aSl, Xa Bang commune, Chau Duc DiStrict , Ba Ria—Vung Tau Province, 25 October 2016, leg. Truong Thuy Ai . PARATYPES: 2 matureS (CTU-EW.166.p02) Same data aS for holotype; 1 mature (CTU- EW.166.p03) Anacardium occidentale plantation (10o39’42.7 N; 107o09’23.0 E), 107 m aSl, Lang Lon commune, Chau Duc DiStrict , Ba Ria—Vung Tau Province, 24 October 2016, leg. Truong Thuy Ai ; 1 mature (CTU- EW.166.p04), banana plantation (10o38’25.3 N; 107o21’00 E), 62 m aSl, Son Binh commune, Chau Duc DiStrict , Ba Ria—Vung Tau Province, 25 October 2016, leg. Truong Thuy Ai ; 1 mature (CTU-EW.166.p05), Anacardium occidentale plantation (10o38’38.0 N; 107o06’50.0 E), 62 m aSl, Hac Dich commune, Tan Thanh DiStrict, Ba Ria— Vung Tau Province, 24 October 2016, leg. Nguyen Phuc Hau ; 2 matureS (CTU-EW.166.p06), near the road (10o29’09.2 N; 107o10’54.6 E), 75 m aSl, Hac Dich commune, Tan Thanh DiStrict , Ba Ria—Vung Tau Province, 24 October 2016, leg. Nguyen Phuc Hau ; 1 mature (CTU-EW.166.p07), on the way to the Bao Quan Mountain (10o35’39.2 N; 107o07’24.0 E), 59 m aSl, Toc Tien commune, Tan Thanh DiStrict , Ba Ria—Vung Tau Province, 24 October 2016, leg. Nguyen Phuc Hau .

Diagnosis. Medium Size, length 132–169 mm, diameter 4.1–6.1 mm, weight 1.64–3.39 gr (in formalin), and SegmentS 91–125. Three pairS of Spermathecal poreS in interSegmentS 6/7/8/9. Micronephridia Surrounding duct of ampulla. Copulatory poucheS preSent. Ventral diStance between male poreS about 0.35x body circumference. No genital markingS in both Spermathecal and male regionS. InteSt nal origin at xv; caeca Simple, originating at xxvii. TyphloSole Simple, lamelliform. Lymph glandS from 15/16. Holandric.

Etymology. Named after the province Ba Ria-Vung Tau where the SpecieS iS widely diStributed.

Description. External characters: Body cylindrical; medium Size, length 132–169 mm, diameter 4.1–6.1 mm, weight 1.64–3.39 gr, SegmentS 91–125 (N=9). DorSum greyiSh brown, ventrum paler. ProStomium 1/2 epilobouS (open). FirSt dorSal pore in 11/12 or 12/13. Preclitellar Setae Stouter and SparSer than poStclitellar Setae, 31–49 in viii, 57–82 in xxv, 11–17 Setae between two male porophoreS (N=9); Setal diStance aa = 1.5–2.5ab, zz = 1.5–2zy. Clitellum annular, xiv–xvi, without dorSal poreS and Setae. Female pore Single, in mid-ventral xiv. Three pairS of Spermathecal poreS in interSegmentS 6/7/8/9; ventral diStance between Spermathecal poreS approximately 0.4x body circumference. Male poreS located inSide copulatory poucheS on the Setal ring xviii; ventral diStance between male poreS about 0.35x body circumference. Genital markingS abSent in both Spermathecal and male pore regionS.

Internal characters: Septa 5/6/7/8 thick, 8/9/10 abSent, 10/11/12 /13 Slightly thick. OeSophageal gizzard within viii–x. InteStinal origin at xv; caeca Simple, originating at xxvii, and extending anteriorly to xxiv or xxiii. LaSt heartS in xiii. Pharyngeal micronephridia developed in 5/6/7. TyphloSole Simple, lamelliform. Lymph glandS bag- Shaped, from 15/16. Three pairS of Spermathecae in vii–ix. Ampulla large, egg-Shaped; duct Short, ca. 1/3 ampulla length, Somewhat conStricted in the middle; micronephridia Surrounding duct of ampulla, moSt developed in Spermathecae at 6/7. Diverticulum Shorter than ampulla in Situ, but longer when extended, Strongly folded, directly attached to the middle of ampulla duct; diStal part of diverticulum enlarged to be a Small, oval-Shaped Seminal chamber. AcceSSory glandS 2–3, Small, muSroom-Shaped; duct long, attached to baSe of ampulla. Holandric. TeStiS SacS developed in x and xi, unconnected. Seminal veSicleS well developed in xi–xii. OvarieS inviSible; oviduct well developed after Septum 12/13. ProState glandS deeply lobuled, paired in xvi–xix; proStatic ductS long, Somewhat enlarge in middle, open in large chamber. AcceSSory glandS large and covering copulatory pouch.

DNA barcodes. A 660bp fragment of the mitochondrial gene Cytochrome c oxydaSe Subunit I waS Sequenced from the holotype, and uploaded to GenBank with the acceSSion number MF481211 .

TABLE]. Morphological comparison of P.vungtauensis sp nov. with similar species within the P. dubia species group. All species lack genital markings in the spermathecal anđ male pores region. AG: accessory glanđs. MP: Male pores.

Remarks. The new SpecieS can be keyed to the dubia group in SimS & EaSton (1972) characteriSed by having three pairS of Spermathecal poreS in interSegmentS 6/7/8/9. ThiS group currently conSiStS of ten SpecieS, Pheretima dubia HorSt, 1893 , P. korinchiana Cognetti, 1922 , P. poiana MichaelSen, 1913 , all from IndoneSia; P. philippina RoSa, 1891 , P. callosa GateS, 1937, P. balbalanensis Hong & JameS, 2010 , P. banaoi Hong & JameS, 2010 , P. globosa Hong & JameS, 2011 , P. julkai Hong & JameS, 2011 , P. lamaganensis Hong & JameS, 2011 , all from the PhilippineS. Morphological differenceS among theSe SpecieS are Summarized in Table 1.

Among theSe SpecieS, the new SpecieS iS fairly Similar to Pheretima balbalensis , P. banaoi , P. julkai , and P. lamaganensis , all from the PhilippineS, in having three pairS of Spermathecal poreS in interSegmentS 6/7/8/9, no genital markingS in Spermathecal and male regionS, and inteStinal caeca Simple. Pheretima balbalanensis iS diStinguiShed by inteStine beginning in xvi, caeca from xxviii, typhloSole veStigial, lymph glandS from xxviii, whereaS P. vungtauensis sp. nov. haS inteStine originating at xv, caeca from xvii, typhloSole lamelliform, bag- Shaped lymph glandS from 15/16. The new SpecieS iS different from P. banaoi in larger ventral diStance between maleS porophoreS (0.35x circumference vS. 0.19–0.22x), inteStinal origin (xv vS. xvi), the beginning of lymph glandS (15/16 vS. xxviii), form of typhloSole (lamelliform vS. Simple fold). The new SpecieS alSo differS from P. julkai and Ph. lamaganensis in ventral diStance between male poreS (0.35x circumference vS. 0.2–0.23x or 0.21– 0.22x), poSition of gizzard (viii–ix vS. viii), inteStinal origin (xv vS. xvi), typhloSole (lamelliform vS. abSent).

Morphologically, the new SpecieS iS particularly Similar to Metaphire houlleti ( Perrier, 1872) in body Shape, three pairS of Spermathecal poreS in interSegmentS 6/7/8/9, preSence of copulatory poucheS, Shape of Spermathecal ampulla and diverticulum, and preSence of aSSeSSory glandS in Spermathecal and male regionS ( Perrier 1872). However, Pheretima vungtauensis sp. nov. iS diStinctly different from M. houlleti in having micronephridia on the Spermathecal ductS, and in the abSence of genital markingS in Spermathecal and male regionS. M. houlleti iS recogniSed by abSence of micronephridia on Spermathecal ductS, and preSence of Small genital markingS inSide Spermathecal and maleS poreS (Blakemore 2016).

Molecular comparison. Due to theSe morphological SimilaritieS, the COI SequenceS from SpecimenS identified aS M. houlleti were included in the molecular analySiS. BaSed on COI SequenceS, the new SpecieS, Pheretima vungtauensis sp. nov., iS more cloSely related to M. houlleti than to other SpecieS of Pheretima ; the holotype of the new SpecieS iS neSted within a clade formed by SpecimenS identified aS Metaphire houlleti ( Fig. 2 ). It iS cloSely related to a Metaphire houlleti group from Thailand (II) with bootStrap and BI valueS of 88% and 1.00 BPP, reSpectively. However, the Kimura 2-parameter (K2P) diStance between the new SpecieS and Metaphire houlleti from Thailand (II) varieS from 0.148 to 0.2 ( Table 2). ThiS diStance iS relatively cloSe to or higher than that for SpecieS delimination uSing COI barcodeS (0.15) aS diScuSSed in Chang & JameS (2011) and Jerathitikul et al. (2017).

In addition, Metaphire houlleti nominal SpecieS iS branched into three cladeS, Vietnam + India + Philippine, Thailand (I) and Thailand (II). Each clade iS clearly different from otherS in genetic diStance (ranging from 0.175 to 0.222). Of theSe, two cladeS ( Thailand I and II) are Separated with good Support of bootStrap and BI valueS of 70% and 0.99 BPP, reSpectively. BeSideS, Blakemore (2016) reportS on a 19% COI difference between two SpecimenS of M. houlleti , one from Thailand, the other one from the PhilippineS, indicating a "molecular SpecieS-group" (ibd.). Furthermore, Jeratthitikul et al. (2017) alSo Stated that M. houlleti nominal SpecieS containS Several new cryptic SpecieS. We agree that the nominal SpecieS M. houlleti needS to be reviSed baSed on SpecimenS collected from different regionS.

Figures

FIGURE 1. Pheretima vungtauensis sp. nov., hOlOtype. A, I: Ventral vieW OF male regiOn (mp = Opening OF cOpulatOry pOuch). B, H, K. Spermathecae (amp = ampulla; dv = diverticulum; ag = accessOry gland; nph = micrOnephridia). C, F. PrOstatic gland (ag = accessOry gland). D, J. Ventral vieW OF spermathecal regiOn (sp = spermathecal pOre). E, G. Intestinal caecum. Scale bar = 1 mm.

FIGURE 2. PhylOgenetic tree inFerring FrOm a dataset OF 523 bp Fragment OF COI using MaXimum LikelihOOd and Bayesian InFerence analysis. BOOtstrap and BI values are shOWn at nOde; # are values less than 65% (ML) Or 0.7 BPP. Sequences With KU are FrOm Jeratthitikul et al. (2017); With KT are FrOm BlakemOre (2016); With MF are FrOm this study; With LC are FrOm Aspe et al. (2016); With KX are FrOm GenBank (unpublished). TWO spequences (MF481209, MF481210) are FrOm samples cOllected FrOm SOc Trang PrOvinve (CTU-EW.DNA.006.01) and Ba Ria—Vung Tau PrOvince (CTU-EW.DNA.006.02).