Morphology and molecular phylogeny of Macrobrachium prachuapense sp. nov. (Decapoda: Palaemonidae) from Southern Thailand Author Saengphan, Nukul 0000-0003-1949-252X Suphanburi College of Agriculture and Technology, Institute of Vocational Education in Agriculture Central Region, Dan Chang, Suphan Buri, Thailand & nukul _ sae @ hotmail. com; https: // orcid. org / 0000 - 0003 - 1949 - 252 X nukul_sae@hotmail.com Author Panijpan, Bhinyo Faculty of Science, Mahidol University, 272 Rama 6 Road, Phayathai, Bangkok 10400, Thailand Author Senapin, Saengchan 0000-0002-5599-4343 National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, Thailand & Center of Excellence for Shrimp Molecular Biology and Biotechnology, Mahidol University, Bangkok, Thailand & saengchan @ biotec. or. th; https: // orcid. org / 0000 - 0002 - 5599 - 4343 saengchan@biotec.or.th Author Laosinchai, Parames Institute for Innovative Learning, Mahidol University, Salaya, Nakhon Pathom, Thailand. Author Suksomnit, Auaree 0000-0003-3553-8567 Suphanburi College of Agriculture and Technology, Institute of Vocational Education in Agriculture Central Region, Dan Chang, Suphan Buri, Thailand & fairyshrimp 1966 @ gmail. com; https: // orcid. org / 0000 - 0003 - 3553 - 8567 fairyshrimp1966@gmail.com Author Phiwsaiya, Kornsunee 0000-0003-3876-6450 National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, Thailand & Center of Excellence for Shrimp Molecular Biology and Biotechnology, Mahidol University, Bangkok, Thailand & kornsunee @ yahoo. com; https: // orcid. org / 0000 - 0003 - 3876 - 6450 kornsunee@yahoo.com text Zootaxa 2021 2021-05-05 4966 4 428 442 journal article 6503 10.11646/zootaxa.4966.4.2 be5233d5-98c2-44f3-bea4-bc9fa14ae4f3 1175-5326 4738718 18AFCD0D-DC50-49A2-9FFF-1E2C61F02D90 Macrobrachium prachuapense Saengphan & Panijpan , sp. nov. ( Figs. 2 – 4 ) Material examined. Holotype : male (carapace length (CL) 8.8 mm ), 6 April 2018 , Khao Chao subdistrict, Pran Buri district , Prachuap Khiri Khan Province ( 12°20.898′ N , 99°41.829′ E ) . Paratypes : 7 males (CL 5.9–9.4 mm ) and 11 females (5.3–9.0 mm), same location and date as holotype . Others : 4 males ( 6.1–9.2 mm ) and 5 females ( 5.5–7.8 mm ), 7 April 2018 , Huai Mae Phriang subdistrict, Kaeng Krachan district , Petchaburi Province ( 12°57.565′ N , 99°24.101′ E ). The holotype (THNHM-Iv-18710) and some paratype specimens (THNHM-Iv-18711, THNHM-Iv- 18712, and THNHM-Iv-18713) of the described species were deposited at the Thailand Natural History Museum . Other specimens were kept at Suphanburi College of Agriculture and Technology, the workplace of the first author . Largest male: total length 42.0 mm, carapace length 9.4 mm (fig. 2) FIGURE 2. Macrobrachium prachuapense sp. nov. male ( upper ) and female ( lower). Largest ovigerous female: total length 39.0 mm, carapace length 9.0 mm. Description. ( Fig. 3A ) Rostrum: straight, dorsal margin convex, extending slightly beyond end of antennal peduncle, subequal to half of carapace length; upper part having 11–15 teeth (usually 11–12), 3–4 teeth behind post orbital margin; lower part having 1–4 teeth (usually 2). Carapace: smooth with fine spinules at antero-ventral surface; antennal spine well developed with pointed end and situated below orbital angle (suborbital angle); hepatic spine smaller than antennal spine and located slightly behind and lower than antennal spine; paired hepatic spines situated between upper first and third teeth of rostrum on carapace. Abdomen: fourth thoracic sternite without median process; sixth abdominal somite 1.40–1.53 times as long as fifth; first, second, and third abdominal sternites having transverse ridges with median teeth only on first and second; preanal region with rounded ridge. Telson: upper telson (fig. 3B) smooth and 1.52–2.20 times length of sixth segment with 2 dorsal paired spines; tail tapering toward rear with pointed end (median spine), each side flanked by 2 spines, outer lateral spine and inner intermediate spine; several pairs of plumose setae found between 2 flanking spines; lateral spines similar in size to dorsal spines; well-developed intermediate spines 2–3 times length of median spine. Eyes: well developed with cornea diameter as long as stalk. Basal segment of antennal peduncle: broad, stylocerite distinctly pointed, reaching to about one-third of basal segment; anterolateral tooth reaching about middle of second segment; second segment as long as third. Scaphocerite (fig. 3C): length 2.78 times width, outer lateral side straight. Epistome (fig. 3D): trilobed by deep depression, but occasionally anteromedian lobe indistinct. Mandibular palp (fig. 3E): slender with three segments, incisor process robust. Maxillular palp (fig. 3F): bilobed, upper lobe stout with apical and subapical setae, lower lobe stout with distal blunt knob bearing short sinuous apical seta. Maxillary palp (fig. 3G): simple without distal setae, basal endite deeply bilobed, scaphognathite normal. First maxilliped (fig. 3H): palp with apical setae, basal and coxal endites distinct, flagellum of exopod with numerous distal plumose setae, epipod bilobed. Second maxilliped (fig. 3I): with normal endopod, flagellum with numerous distal setae, epipod simple. Third maxilliped (fig. 3J): with robust endopod, exopod with numerous distal plumose setae, reaching up to distal margin of ischiomerus. First pereiopod (fig. 3K): slender, end of carpus reaching end of scaphocerite, equal in length, similar in form; palm as long as finger, carpus 1.80 times as long as chela, merus shorter than carpus. FIGURE 3. Macrobrachium prachuapense sp. nov. (male). (A) Entire animal, lateral view; (B) telson; (C) scaphocerite; (D) epistome; (E) mandible; (F) maxillula; (G) maxilla; (H) first maxilliped; (I) second maxilliped; (J) third maxilliped; (K) first pereiopod; (L) third pereiopod; (M) movable spine. Scales: (A) 5 mm; (C, J) 2 mm; (D, M) 0.25 mm; (F) 0.5 mm; (K, L) 3 mm; others 1 mm. Second pereiopod (fig. 4): distinctly unequal in length but similar in shape; major second pereiopod reaching beyond scaphocerite by end of merus, as long as or prolonging to noticeably longer than total length; palm, finger, and on proximal ½–4/5 on dorsal and ventral parts of carpus covered with long velvet setae; merus not inflated, 4.25 times as long as height, subequal in length to palm, covered with numerous spinules over its length and sparsely with long velvet setae on certain spots; carpus elongated and subcylindrical, 2.75 times as long as height, with numerous spinules over its length; palm comparatively slender, subequal or shorter than fingers, 3.2 times as long as width, on proximal 1/5 of both lateral sides covered with spinules; fingers equal or longer than palm, 15–28 teeth on cutting edges, curved inwards, tips crossing and small gaping when closed. FIGURE 4. Ventral view of major second pereiopod (upper) and its chela (lower) of male Macrobrachium prachuapense sp. nov. Minor second pereiopod: short, about 0.74–0.9 times of total length; fingers 1.3–2.0 times as long as palm, with numerous teeth (13–15 teeth) on proximal ¾–4/5 of both cutting edges; merus 1.5–1.6 times as long as carpus; palm subequal to carpus, 2.7 times as long as wide, with setae as on major second pereiopod. Last three pereiopods: slender, similar in form; propodus, carpus, and merus covered with spinules and setae. Third pereiopod (fig. 3L): reaching or nearly so to end of scaphocerite; propodus 2.72–3.08 times as long as dactylus with 7 movable spines on postero-lateral margin. Fifth pereiopod: somewhat slenderer than third, reaching or nearly so to distal margin of scaphocerite. First pleopod: endopod not reaching distal half of exopod, inner margin concave, outer margin slightly convex. Appendix musculina of male: longer and stouter than appendix interna with numerous stiff setae. Uropodal diaeresis: with inner movable spine (fig. 3M) longer than outer angle. Ovigerous females: with eyed eggs 1.10×1.45 – 1.28× 1.75 mm in diameter, 25–45 per ovigerous female. Etymology. The specific name “ prachuapense ” refers to the collection site of the holotype , Prachuap Khiri Khan Province , which is commonly called Prachuap. Molecular phylogeny. DNA sequences: COI 667 base pairs (bp), consensus alignment 678 bp; 18S 1446 bp, consensus alignment 1458 bp. The COI alignment contained 293 variable sites, 245 of which were informative. The 18S alignment contained 127 variable sites, 62 of which were informative. However, only five of these 62 informative sites were useful for phylogenetic analysis within the Macrobrachium pilimanus species group. Genetic distances: Table 3 shows the pairwise p -distances among the COI sequences of Macrobrachium prachuapense sp. nov. and M. naiyanetri . It clearly indicates that M. prachuapense sp. nov. is significantly different from M. naiyanetri from the southern part of Thailand from where the holotype was collected. Table 4 shows the average interspecific p -distances among the COI sequences of the clades containing multiple closely-related sequences (pairwise p -distances <8%). Table 5 shows the average intraspecific p -distance among the COI sequences within each clade. The clades in Tables 4 and 5 are based on the phylogenetic relationship in fig. 5. TABLE 3. Pairwise p -distances among the COI sequences of Macrobrachium prachuapense sp. nov. and all the M. naiyanetri . The headers refer to the six digits of the sequences’ GenBank accession numbers (Table 2). Only the first sequence belongs to M. prachuapense sp. nov. Other sequences are arranged by their distances to the first in ascending order. The lower left part represents the p -distances while the upper right one refers to their standard errors of estimates.

Acc. no.	459320	235960	235951	235967	235968	235954	235955
459320	0.0040	0.0061	0.0098	0.0098	0.0099	0.0103
235960	0.0120	0.0055	0.0098	0.0098	0.0098	0.0103
235951	0.0270	0.0221	0.0103	0.0103	0.0103	0.0103
235967	0.0705	0.0678	0.0723	0.0015	0.0015	0.0036
235968	0.0720	0.0693	0.0737	0.0015	0.0022	0.0039
235954	0.0720	0.0693	0.0737	0.0015	0.0029	0.0039
235955	0.0765	0.0737	0.0752	0.0088	0.0103	0.0103

FIGURE 5. Phylogenetic tree containing Macrobrachium prachuapense sp. nov. reconstructed from mitochondrial COI and nuclear 18S genes using partitioned Bayesian inference with Cryphiops caementarius and Coralliocaris superba constituting an outgroup. Each leaf taxon is indicated by its scientific name and COI accession number (Table 2). The letters and numbers at the nodes represent the clade names and posterior probabilities supporting the corresponding clades respectively. Branch lengths represent the estimated numbers of nucleotide substitutions per site. TABLE 4. Average interspecific p -distances among the COI sequences of the clades containing multiple closely-related sequences (pairwise p -distances <8%). The headers refer to the clades in fig. 5. The lower left part represents the p -distances while the upper right one refers to their standard errors of estimates.

Clade	A	B	C	D	E	F	G	H	I	J	K	L	M	N	O
A	0.0092	0.0103	0.0111	0.0118	0.0117	0.0114	0.0122	0.0112	0.0124	0.0139	0.0149	0.0145	0.0147	0.0141
B	0.0722	0.0110	0.0116	0.0120	0.0118	0.0120	0.0123	0.0111	0.0124	0.0129	0.0143	0.0137	0.0145	0.0136
C	0.0855	0.0906	0.0118	0.0120	0.0118	0.0121	0.0122	0.0115	0.0129	0.0136	0.0150	0.0139	0.0145	0.0142
D	0.1031	0.1067	0.1050	0.0111	0.0117	0.0121	0.0128	0.0112	0.0124	0.0136	0.0147	0.0140	0.0143	0.0144
E	0.1112	0.1110	0.1106	0.1037	0.0118	0.0121	0.0122	0.0115	0.0124	0.0135	0.0146	0.0141	0.0145	0.0143
F	0.1149	0.1066	0.1123	0.1136	0.1062	0.0116	0.0123	0.0111	0.0124	0.0139	0.0150	0.0142	0.0144	0.0141
G	0.1153	0.1203	0.1155	0.1252	0.1162	0.1131	0.0096	0.0113	0.0120	0.0130	0.0153	0.0145	0.0136	0.0138
H	0.1234	0.1238	0.1156	0.1362	0.1217	0.1284	0.0845	0.0109	0.0113	0.0132	0.0148	0.0140	0.0146	0.0144
I	0.1085	0.0963	0.1122	0.1082	0.1080	0.1013	0.1076	0.1073	0.0109	0.0134	0.0144	0.0139	0.0145	0.0140
J	0.1313	01285	0.1437	0.1361	0.1338	0.1372	0.1324	0.1274	0.1069	0.0144	0.0147	0.0135	0.0141	0.0143
K	0.1495	0.1324	0.1453	0.1438	0.1468	0.1475	0.1471	0.1459	0.1403	0.1777	0.0154	0.0142	0.0144	0.0136
L	0.1970	0.1712	0.1896	0.1821	0.1886	0.1926	0.2039	0.1997	0.1828	0.1977	0.2048	0.0107	0.0144	0.0138
M	0.2012	0.1784	0.1868	0.1945	0.1903	0.1883	0.2025	0.1958	0.1819	0.2012	0.1946	0.1107	0.0132	0.0129
N	0.1807	0.1704	0.1742	0.1747	0.1841	0.1866	0.1838	0.1963	0.1758	0.1794	0.1791	0.1806	0.1805	0.0114
O	0.1918	0.1774	0.1914	0.1887	0.1897	0.1976	0.2002	0.2013	0.1896	0.1928	0.1699	0.1876	0.1837	0.1163

Phylogenetic relationship (fig. 5): Based on the combined (COI and 18S) sequences, Macrobrachium prachuapense sp. nov. is novel and different from the other closely related species. The novel prawn and M. naiyanetri with COI accession numbers MT235951 and MT235960 form monophyletic clade (clade A), posterior probability 1; clade A and other M. naiyanetri (clade B) form monophyletic clade (clade P), posterior probability 0.94; clade P and M. forcipatum (clade C) form monophyletic clade (clade Q), posterior probability 1; clade Q and M. palmopilosum Siriwut et al. , 2020 (clade D) form monophyletic clade (clade R), posterior probability 0.76; clade R and M. malayanum with COI accession numbers MT235965 –6 (clade E) form monophyletic clade (clade S), posterior probability 0.59; M. puberimanus Siriwut et al. , 2020 and M. sp. ( cf. dienbienphuense ) form monophyletic clade (clade H), posterior probability 1; clade H and M. dienbienphuense (clade G) form monophyletic clade (clade T), posterior probability 1; clade T, M. hirsutimanus ( Tiwari, 1952 ) (clade I), and M. eriocheirum (clade J) form monophyletic clade (clade U), posterior probability 1; clades S and U and M. malayanum with COI accession numbers MT235959 and MT235964 (clade F) form monophyletic clade (clade V), posterior probability 1; clade V and M. sirindhorn Naiyanetr, 2001 (clade K) form monophyletic clade (clade W), posterior probability 1; M. niphanae Shokita & Takeda, 1989 (clade L) and M. chainatense Saengphan et al ., 2019 (clade M) form monophyletic clade (clade X), posterior probability 1; clades W and X form monophyletic clade (clade Y), posterior probability 1; clade Y and M. saengphani Saengphan et al ., 2020 form monophyletic clade (clade Z), posterior probability 0.56; M. sintangense ( de Man, 1898 ) (clade N) and M. suphanense Saengphan et al ., 2018 (clade O) form monophyletic clade (clade AA), posterior probability 1; M. lanchesteri ( de Man, 1911 ) and M. rosenbergii ( de Man, 1879 ) form monophyletic clade (clade AB), posterior probability 1; clades AA and AB form monophyletic clade (clade AC), posterior probability 0.57; clades Z and AC are sisters, posterior probability 1.