﻿Molecular phylogeny and taxonomic position of Macrobrachiumlanchesteri (De Man, 1911), with descriptions of two new species from Thailand (Decapoda, Caridea, Palaemonidae)

﻿Abstract Macrobrachiumlanchesteri (De Man, 1911), a translucent freshwater prawn has a wide distribution range throughout mainland Southeast Asia. A high morphological variation and genetic divergence between different geographical M.lanchesteri populations in Thailand have peculiarly extended the uncertainty of species boundaries and blended confusingly with several Macrobrachium species. To clarify these circumstances, broad sample examinations of the morphological variation, including topotype specimens, and phylogenetic reconstruction based on the concatenated mitochondrial dataset (16s rRNA and COI genes) were performed. Broad morphological examination of M.lanchesteri has shown congruency with phylogenetic analyses by revealing prominent lineages of M.lanchesteri sensu stricto and two new sibling lineages with interspecific variation between 6.48–8.76% for COI and 3.06–4.23% for 16S. Descriptions of two new species, named herein as M.panhai Chaowvieng & Siriwut, sp. nov. and M.rostrolevatus Chaowvieng & Siriwut, sp. nov. are provided. Morphological investigation of rostral form suggested plasticity in M.rostrolevatus populations showing the morphological trait associated with their habitat preferences. Furthermore, phylogenetic positions of the three taxa affirmed the hidden diversity of Thai freshwater Macrobrachium fauna correlated with the river network in the Mekong and Chao Phraya basins, Thailand. The genetic data and distribution records obtained in this study may also assist future river conservation plans as well as the sustainable management of freshwater prawn diversity.


Introduction
Palaemonid freshwater prawns of genus Macrobrachium Spence Bate, 1868 have shown high species richness comprising 271 species worldwide (WoRMS 2023).This genus has a broad geographical distribution and is commonly found in the Oriental Region of Asia (De Grave et al. 2008).Several Macrobrachium ZooKeys 1190: 163-193 (2024), DOI: 10.3897/zookeys.1190.113898 Apisara Chaowvieng et al.: Molecular phylogeny and morphology of two Macrobrachium prawns from Thailand species demonstrate economic impacts, serving as protein resources and for utilisation in ornamental fish aquaculture (Cai et al. 2004;Wowor et al. 2004).According to its remarkable species richness and diversifications of aquatic and terrestrial invertebrate faunas in Indochina, the intensive fauna exploration and historical biogeography using both morphology and genetics were reinvestigated systematically in several taxa such as river prawns, bivalves, land snails and millipedes (De Bruyn et al. 2014;Pholyotha et al. 2021;Jeratthitikul et al. 2022;Likhitrakarn et al. 2023).Indochinese Macrobrachium prawns have gained attention recently, especially in the context of taxonomy and systematics (Cai et al. 2004;Wowor et al. 2004;Hanamura et al. 2011).Several molecular taxonomic studies have been verified nominal species and consequently supplemented the taxonomic account of some cryptic Macrobrachium prawns (Mar et al. 2018;Siriwut et al. 2020;Jurniati et al. 2021;Saengphan et al. 2021).Additionally, the DNA barcoding and molecular delimitation methods were implemented to clarify the taxonomic boundaries of several Macrobrachium species.Moreover, the phylogenetic positions of several species have been addressed some morphological complexity groups based on barcode gap distance threshold (Siriwut et al. 2021).
Currently, 34 species have been documented in Thailand (Cai et al. 2004;Cai and Vidthayanon 2016;Saengphan et al. 2018Saengphan et al. , 2019Saengphan et al. , 2020Saengphan et al. , 2021;;Siriwut et al. 2020Siriwut et al. , 2021)).Two major river basins, the Chao Phraya and the Greater Mekong, have been discussed as being significant hotspots for native Macrobrachium faunal diversity (Cai and Ng 2002;Hanamura et al. 2011).Some Thai Macrobrachium species have been reported to show narrow distribution within these basins, such as M. chainatense Saengphan, Panijpan, Senapin, Laosinchai, Ruenwongsa, Suksomnit & Phiwsaiya, 2019 which was only found in Central Thailand, and M. spelaeus Cai & Vidthayanon, 2016 that live in stygobiotic habitats.Contrastingly, some widespread species have also been documented about their distribution occupancy crossed inland basins and some insular territory of Southeast Asia, such as M. sintangense (De Man, 1898), and M. dienbienphuense Dang & Nguyen, 1972(Cai et al. 2004;Wowor et al. 2004;Hanamura et al. 2011).For this reason, freshwater faunas in Thailand and neighbouring countries are capable linkage in terms of species composition, reaching an occurrence data of coexistence and cryptic species according to the connection of the river network (Hanamura et al. 2011;Siriwut et al. 2020).
A small translucent and common M. lanchesteri (De Man, 1911) dominantly occupies all river basins throughout mainland Southeast Asia with scattered distribution records from Malaysia, Singapore, Indonesia; it has even expanded northward to South China (Wowor and Choy 2001;Cai and Ng 2002;Cai et al. 2004).This species was originally found in southern Thailand and was diagnosed as having a straight and short rostrum not exceeding the scaphocerite and slender, thin second pereiopods (Kemp 1918;Holthuis 1950).The lectotype designation and morphological study of M. lanchesteri by Chong and Khoo (1988) advocated diagnostic character variation, particularly on rostral structure and body size variation in male regarding sexual dimorphism.Additionally, M. lanchesteri was mentioned with an argument on taxonomic boundary with some other congeners such as M. peguense (Tiwari, 1952), M. kistnense (Tiwari, 1952), and M. tiwarii Jalihal, Shenoy & Sankolli, 1988.Moreover, M. lanchesteri also blended confusingly with the juveniles of several species such as M. idae (Heller, 1862) and M. lar (Fabricius, 1798) (Lanchester 1902;Kamita 1966).
Previous phylogenetic and population genetic studies of M. lanchesteri in Thailand have also detected high genetic diversity, both between and within populations (Reingchai et al. 2009;Khanarnpai et al. 2019;Siriwut et al. 2021).Moreover, the possible existence of cryptic species within several Macrobrachium species in Thailand under traditional morphological discrimination criteria was reported, including M. lanchesteri, based on DNA barcode delimitation thresholds (Siriwut et al. 2021).The lack of intensive collection from different river basins impeded comprehensive genetic and morphological information that would contribute to taxonomic boundary clarification and phylogenetic relationships of M. lanchesteri and other native species in this area.To elucidate the taxonomic confusion of several coexistent translucent Macrobrachium prawns, the integration of traditional morphological identification and molecular analysis could be investigated concurrently.Therefore, this study aimed to clarify the taxonomic boundaries of M. lanchesteri in Thailand by broad-scale sampling and reconstruct the phylogenetic relationships with various related translucent species based on COI gene and 16S rRNA markers, which have been used extensively to investigate the phylogenetic relationships between crustaceans (Costa et al. 2007;Pileggi and Mantelatto 2010;Castelin et al. 2017;Jamaluddin et al. 2019;Rossi et al. 2020).This study will contribute to elucidate the taxonomic status of M. lanchesteri s. str.and its closely related species as well as assist economical freshwater prawn management in the future.

Sample collection and preparation
Prawn specimens were collected from various freshwater basins in Thailand.Live specimens were photographed to document body coloration using a Nikon D5300 camera with a micro-Nikkor 105 mm f/2.8 IF-ED Macro Lens.Prawns were gradually euthanised following the protocols approved by the Mahidol University-Institute Animal Care and Use Committee (MU-IACUC) under approval number MUSC66-026-656.Specimens were preserved in 95% ethanol and stored into a container for further morphological examination and molecular analysis.Voucher specimens were deposited at the Chulalongkorn University Museum of Zoology, Bangkok, Thailand (CUMZ) and Mahidol University Museum of Natural History, Department of Biology, Faculty of Science, Mahidol University, Thailand (MUMNH).Traditional identifications were carried out based on previous taxonomic studies of Macrobrachium species: Lanchester (1902), Holthuis (1950), Chong and Khoo (1988), Cai and Ng (2002), Cai et al. (2004), Wowor et al. (2004), andHanamura et al. (2011).The morphological variation of prawn specimens was observed and illustrated under a stereomicroscope.A list of abbreviations used in the descriptions is given as follows: Fin (finger), Pal (palm), Car (carpus), Mer (merus), Che (chela), Dac (dactylus), Pro (propodus), cl (carapace length), rl (rostrum length).All morphological characters were measured using Dinocapture software v. 2.0 and reported in millimetres.

DNA extraction and PCR protocol
All prawn specimens used for molecular analysis in this study are listed in Table 1.Genomic DNA was extracted from pleonal muscle tissue by using DNA extraction kits (NucleoSpin Tissue kit: MACHEREY-NAGEL).Genomic DNA quality was evaluated and visualised by gel electrophoresis and a UV illuminator.Two mitochondrial genes, 16S rRNA and cytochrome c oxidase subunit I (COI), were amplified.Three sets of primer such as 16Sa-L (5' CGC CTG TTT ATC AAA AAC AT 3') and 16Sbr-H2 (5' CTC CGG TTT GAA CTC AGA TCA 3') following Palumbi (1996) for 16S gene, LCO1490 (5'GGT CAA CAA ATC ATA AAG ATA TTG G 3'; Folmer et al. (1994), MacroNancy (5' GCG GGT AGR ATT AAR ATR TAT ACT TC 3'; Siriwut et al. (2020), HCOoutout (5' GTA AAT ATA TGR TGD GCTC 3';Schulmeister et al. (2002) for COI were used in this study.PCR was performed using T100 TM thermal cycler (BIO-RAD) with a gradient temperature function.The PCR profile consisted of the following steps: 94 °C for 5 min as an initial step followed by 34 cycles 94 °C for 30 sec for denaturing, 45-49 °C for 40 sec, 72 °C for 15 sec for extension, and final extension at 72 °C for 10 min.PCR products were run by 1% agarose gel electrophoresis stained with SYBR Safe illuminant (Invitrogen, USA).The purified products were sent for sequencing by a commercial company (Macrogen and Bioneer, Korea) using an Applied Biosystems automatic sequencer.

Phylogenetic analyses
Sequences were aligned and corrected using the ClustalW algorithm in MEGA 11 (Tamura et al. 2021).All sequences have been registered and deposited in Gen-Bank database under accession numbers OR575072-OR575118 for COI and OR578642-OR578698 for 16S (Table 1).The voucher specimen locality of each species used in molecular analysis is illustrated in Fig. 1.The DNA dataset for phylogenetic analyses was assembled including ten deposited COI sequences of Macrobrachium species in GenBank database.To depict the clade of M. lanchesteri sensu De Man (1911), topotype sequences were selected as representative indicators.Macrobrachium villosimanus (Tiwari, 1949) was used as the rooting outgroup.
Phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI) methods throughout the online CIPRES Science Gateway server (Miller et al. 2010).The concatenated dataset of two markers with the partitioned file for nucleotide substitution model fit was prepared in Kakusan 4 (Tanabe 2007).ML tree was visualised in RAxML v. 8.2.12.(Stamatakis 2014).The GTR+G model was set as the model for all gene partitions with 1,000 bootstrap replicates performed to verify tree topology and clade support.BI tree was estimated using MrBayes v. 3.2.7 (Ronquist et al. 2012).Markov chain Monte Carlo (MCMC) was configured as 10,000,000 generations of the sampling process; the first 25% of obtained trees were discarded as burn-in.Finalised trees were estimated for the consensus tree topology.The annotation and illustration of clade and branch length were performed in Figtree (Rambaut 2010).Node posterior probabilities of 0.95 were considered statistically significant for BI, and bootstrap support values greater than 70 were considered highly supported for ML (Huelsenbeck and Hillis 1993;Larget and Simon 1999).Pairwise genetic distance of intra and interspecific of each gene dataset was calculated using the p-distance method in MEGA 11 (Tamura et al. 2021).

Molecular phylogeny and genetic divergence
Forty-seven sequences of partial COI and 57 sequences of partial 16S genes were successfully amplified and obtained (Table 1).COI sequence contained 627 bp with 417 bp of conserved sites, 210 bp of variable sites and 202 of parsimony informative sites.16S sequence contained 554 bp with 373 bp of conserved sites, 181 bp of variable sites and 154 bp of parsimony informative sites.The proportional range of genetic variations in M. lanchesteri species complex and other Macrobrachium species were revealed by p-distance.Inter and intraspecific variations ranged from 15.12-20.68%for COI, 8.6-16.18%for 16S and 0.9-5.79%for COI and 1.08-3.19%for 16S, respectively.Both ML and BI trees based on 1,181 bp concatenated dataset of the COI and 16S gene fragments revealed the six Macrobrachium species as monophyletic groups with strong statistical support values (Fig. 2).Clade C comprised all M. sintangense sequences.Phylogenetic tree also showed that M. rosenbergii (De Man, 1879) is closely related to M. lanchesteri species complex clade, forming clade D. The genetic distance between M. rosenbergii and M. lanchesteri species complex clade was 15.12% for COI and 8.6% for 16S.In the clade E, Macrobrachium lanchesteri species complex was divided into three monophyletic groups with high statistical supports for both ML (100) and BI (1).The interspecific variation ranged from 6.48-8.76%for COI and 3.06-4.23%for 16S.The intraspecific variation also ranged from 0.92-2.27%for COI and 0.7-2.23%for 16S.In the results of this study, clade H was shown as M. lanchesteri based on the topotype sequences assembled.The monophyletic group of M. lanchesteri s. str.herein represents two subclades, lower Isthmus of Kra (Clade I) and upper Isthmus of Kra populations.Macrobrachium panhai sp.nov.(Clade J) was nested as a sister clade of M. lanchesteri s. str.with sufficient support in ML ( 74), but partial support in BI (86).Macrobrachium rostrolevatus sp.nov.(Clade F) was separated from congeneric members of M. lanchesteri species complex and all samples in this clade were strictly distributed inside freshwater basins on the Khorat Plateau, i.e. the Mun, Chi and Songkhram Rivers.
Composite description.Rostrum (Fig. 4B).Straight or slightly convex proximally and upward distally.Rostrum length exceeding end of antennular peduncle and slightly shorter than scaphocerite.Dorsal margin with 6-10 teeth including 1-3 teeth distally with small gap from rest.Postorbital margin with one or two teeth, reaching to one-fourth of carapace length.Ventral margin with 1-6 teeth, starting from middle to distal margins.Short setae present between rostral teeth.
Cephalon (Fig. 4B).Well-developed eye.Ocular beak without laterally expanded tip.Cornea longer and broader than stalk.Postantennular carapace margin rounded.Cornea osculum longer than stalk.Antennular peduncle longer than wide with fine setae, basal segment short, second segment shorter than third segment.Stylocerite projection sharp, reaching beyond basal segment.Antennal spine sharp situated below orbital margin.Hepatic spine slightly larger than antennal spine, positioned posteriorly and lower than antennal spine.Scaphocerite with straight margin, distolateral tooth sharp and not reaching end of lamella.Epistome bilobed (Fig. 4C).Branchiostegal suture starting from carapace margin to behind hepatic spine.Carapace surface smooth.
First pereiopods.Long and slender, reaching end of scaphocerite.Fingers as long as palm, tips with fine setae.Series of setae present at anterior inner part of palm.Carpus slightly longer than merus.Distal articulation of carpus with series of fine setae.Ischium shorter than merus.Scattered setae present on all segments.
Second pereiopods (Fig. 4D).Long and slender, similar in form and exceeding scaphocerite.Fingers subcylindrical covered with scattered setae.Palm 1.1-1.4×longer than fingers.Fingers with translucent razor edge present anteriorly and one or two tiny teeth on proximal quarter of cutting edges.Tip of fingers crossed and covered by fine setae (Fig. 4E).Carpus cylindrical shape and articulation margin expanded.Carpus 1.3-1.5×longer than chela.Merus subcylindrical.Carpus 1.1-2× longer than merus.Scattered short setae present on all segments.
Fourth and fifth pereiopods.Long and slender, exceeding scaphocerite.Propodus of fourth pereiopods with 5-10 pairs of spines distributed along its length, 2× longer than dactylus.Propodus slightly longer than merus.Ischium shorter than merus.Propodus with fine setae at distal articulation.Scattered short setae present on all segments.Propodus of fifth pereiopods with 7-13 pairs of spines distributed along its length and fine setae at distal articulation.Propodus 2× longer than carpus.Propodus as long as merus.Scattered short setae present on all segments.
Thoracic sternum.Fourth and fifth thoracic sternites with transverse plate.Sixth and seventh thoracic sternites smooth.Eighth thoracic sternite with or without acute median process.
Pleon.Smooth.All pleonal sternites with transverse ridge.First and second pleonal sternites usually with small median process.Third and fourth pleonal sternites smooth.Fifth pleonal sternite with triangular ridge.Preanal carina present, obtuse ridge developed without spine or setae.Ventral margin of pleural tergum with small setae.
Telson (Fig. 4G).Tapered posteriorly, protruding point on middle margin with lateral spines and few fine setae.Inner spines longer than outer spines.Dorsal surface with two pairs of small spines similar in size.
Remarks.The specimen collected in this study generally agrees with the original description in Lanchester (1902), and a subsequent description of the lectotype provided by Chong and Khoo (1988).Previous studies reported that male specimens tended to display the sexual dimorphism with a large body size, tomentose fingers, and minute spinules on all segments (except fingers) of second pereiopods.In this study, only one large male specimen, collected from Loei Province, Thailand, exhibits this characteristic.Typically, both male and female specimens possess fine setae on fingers and scattered setae on surface of second pereiopods.Furthermore, this study also observed two variable characters occurring on the second pereiopods.Firstly, the proportional length and form of second pereiopods were found to be variable in specimens from Krabi population.Their second pereiopods are shown to be prominently long and robust, similar to those of M. sintangense (a common riverine species).The palm margin is laterally inflated and slightly shorter than fingers, and the chela slightly longer than the carpus.Additionally, Chong and Khoo (1988) reported the presence of two tiny teeth on the basal portion of cutting edges of fingers in M. lanchesteri as a diagnostic character.In this study, one or two tiny teeth were present on the cutting edges of fingers and vary among M. lanchesteri populations.Historically, M. lanchesteri was noted to resemble several other species including M. idae, M. peguense (see under remarks of M. panhai sp.nov.), M. sankollii Jalihal, Shenoy & Sankolli, 1988, M. unikarnatakae Jalihal, Shenoy & Sankolli, 1988, and M. sintangense.Further phylogenetic relationships and phylogenetic placement of aforementioned taxa should be tested to elucidate and verify their taxonomic identities.
Macrobrachium lanchesteri has a wide distribution across mainland Southeast Asia and southern China.This species can live in various freshwater ecosystems by inhabiting aquatic vegetation in stagnant freshwater habitats such as ponds, lakes, and paddy fields.Diagnosis.Rostrum straight proximally and slightly upward distally.Rostrum length reaching beyond end of antennular peduncle and exceeding the scaphocerite.Rostral formula: 8-12/3-6 teeth including two or three distal teeth with small gap separate from rest.Carapace smooth.Epistome bilobed.First pereiopods reaching end of scaphocerite.Second pereiopods thin and long, similar in form and equals in length, exceeding scaphocerite.Fingers covered with scattered setae, slightly shorter than palm.Translucent razor edge present anteriorly between fingers and no teeth on inner side of cutting edges.Carpus cylindrical shape and articulation margin expanded.Carpus 1.5× longer than chela.Merus subcylindrical.Carpus 1.5× longer than merus.Third pereiopods thin and long, reaching end of scaphocerite.Dactylus curved distally with short setae.Propodus 2× longer than dactylus.Propodus with three or four pairs of spines and fine setae present scarcely on articulation margin.Propodus 2× longer than carpus.Sixth to eighth thoracic sternites smooth.First and second pleonal sternites with small median process or smooth.Third and fourth pleonal sternites smooth.Fifth pleonal sternite with triangular ridge.Uropodal diaeresis with inner movable spine slightly longer than outer angle.

Macrobrachium panhai
Composite description (holotype in parentheses).Rostrum (Fig. 5B).Straight or proximal convex and slightly distal upward.Rostrum length exceeding end of antennular peduncle and slightly exceeding scaphocerite (rl 7.32 mm).Dorsal margin with 8-12 (10) teeth including two or three (3) teeth distally with small gap from rest.Postorbital margin with one or two (1) teeth, reaching one-third of carapace length.First dorsal tooth positioned slightly behind hepatic spine.Ventral margin with 3-6 (4) teeth, starting from middle to distal margin.Short setae present between rostral teeth.
Cephalon (Fig. 5B).Eye well developed.Ocular beak without laterally expanded tip.Cornea longer and broader than stalk.Postantennular carapace margin rounded.Cornea osculum longer than stalk.Antennular peduncle longer than wide, with fine setae.Basal segment short, second segment shorter than third segment.Stylocerite projection sharp, reaching beyond basal segment.Antennal spine sharp, situated below orbital margin.Hepatic spine slightly larger than antennal spine, positioned posteriorly and lower than antennal spine.Scaphocerite with straight margin, distolateral tooth sharp and not reaching end of lamella.Epistome bilobed (Fig. 5C).Branchiostegal suture beginning at carapace margin to behind hepatic spine.Carapace surface smooth (cl 5.76 mm).
First pereiopods.Thin and long, reaching end of scaphocerite.Fingers as long as palm, tips with fine setae.Series of setae present on anterior inner part of palm.Carpus slightly longer than merus.Distal articulation of carpus with series of fine setae.Ischium shorter than merus.Scattered setae present on all segments.
Third pereiopods (Fig. 5F).Thin and slender, reaching end of scaphocerite.Dactylus short and curved distally.Propodus 2× longer than dactylus.Propodus with three or four pairs of spines along inferior-lateral margin and fine setae at distal articulation, 2× longer than carpus.Ischium shorter than carpus.Scattered short setae present on all segments.
Fourth and fifth pereiopods.Long and slender, exceeding scaphocerite.Propodus of fourth pereiopods with 3-6 ( 4) pairs of spines distributed along its length, 2.5× longer than dactylus.Propodus as long as merus.Ischium shorter than merus.Propodus with fine setae at distal articulation.Scattered short setae present on all segments.Propodus of fifth pereiopods with 4-8 pairs of spines distributed along its length and fine setae at distal articulation.Propodus 2.5× longer than carpus.Propodus as long as merus.Scattered short setae present on all segments.
Thoracic sternum.Fourth and fifth thoracic sternites with moderately transverse plate without median process, and seventh thoracic sternite smooth.Eighth thoracic sternite usually smooth.
Pleon.Smooth.All pleonal sternites with transverse ridges.First and second pleonal sternites with or without small median processes.Third and fourth pleonal sternites smooth.Fifth pleonal sternite with triangular ridge.Preanal carina present, obtuse ridge developed without spine or setae.Ventral margin of pleural tergum with small setae.
Telson (Fig. 5G).Tapered posteriorly, protruding point on middle margin with lateral spines and few fine setae.Inner spines longer than outer spines.Dorsal surface with two pairs of small spines, similar in size.
Uropods (Fig. 5G).Uropodal diaeresis with inner movable spine, as long as or slightly longer than outer angle.Exopods longer than endopods.
Etymology.The specific name panhai is dedicated to Prof. Dr. Somsak Panha, a taxonomist from Faculty of Science, Chulalongkorn University, Thailand well known for his remarkable contributions and endorsement to the study of invertebrate fauna in Thailand.
Distribution.This species is distributed in the Chao Phraya and Mekong River Basins, Thailand.
This new species also differs from M. peguense sensu Tiwari (1952) by processes of rostral formula 8-12/3-6 teeth (vs 6-9/2-4 teeth in M. peguense).Second pereiopods had palms shorter than half of carpus (vs palm slightly more than half of carpus in M. peguense).Propodus of third pereiopods are 2× longer than dactylus (vs 3 in M. peguense).Dorsal surface of telson is without depression (vs longitudinal depression in M. peguense).Movable spine at uropodal diaeresis is slightly longer than outer angle (vs movable spine is shorter in M. peguense).Cai and Ng (2002) also mentioned that the egg size can be used to distinguish M. peguense and M. lanchesteri group (1.15-1.5 × 1.6-2.1 mm and 0.6-0.7 × 0.8-1 mm, respectively).Currently, the distribution range of M. peguense was found only from Myanmar.Diagnosis.Rostrum long and thin, proximal half straight and uplifted distal half.Rostrum length reaching beyond end of antennular peduncle and prominently exceeding scaphocerite.Rostral formula: 6-11/4-9 teeth including 2-4 teeth distally with large gap from rest.Apical teeth usually present with trifid.Carapace smooth.Epistome bilobed.First pereiopods reaching end scaphocerite.Second pereiopods thin and long, similar in form and length, exceeding end of scaphocerite.Fingers covered with scattered setae with translucent razor edge present anteriorly between fingers and one tooth on proximal quarter of cutting edges.Palm 1.25× longer than fingers.Carpus cylindrical shape and articulation margin expanded.Carpus 1.5-2× longer than chela.Merus subcylindrical.Carpus 1.5× longer than merus.Third pereiopods thin and long, slightly exceeding scaphocerite.Dactylus curved distally with short setae.Propodus 2× longer than dactylus.Propodus with 3-6 pairs of spines distributed along its length and fine setae at its articulation.Propodus 2× longer than carpus.Sixth to eighth thoracic sternites smooth.First and second pleonal sternites with small median process.Third and fourth pleonal sternites smooth.Fifth pleonal sternite with triangular ridge.Uropodal diaeresis with inner movable spine slightly longer than outer angle.
Cephalon (Fig. 6B).Eye well developed; ocular beak without laterally expanded tip.Cornea longer and broader than stalk.Postantennular carapace margin rounded.Cornea osculum longer than stalk.Antennular peduncle longer than wide, with fine setae.Basal segment short, second segment being shorter than third segment.Stylocerite projection sharp, reaching beyond basal segment.Antennal spine sharp, situated below orbital margin.Hepatic spine slightly larger than antennal spine, positioned posteriorly and lower than antennal spine.Scaphocerite with straight margin, distolateral tooth sharp and not reaching end of lamella.Epistome bilobed (Fig. 6D).Branchiostegal suture starting from carapace margin to behind hepatic spine.Carapace surface smooth (cl 7.14 mm).
First pereiopods.Long and slender, reaching end of scaphocerite.Fingers as long as palm, tips with fine setae.Series of setae present at anterior inner part of palm.Carpus slightly longer than merus.Distal articulation of carpus with series of fine setae.Ischium shorter than merus.Scattered setae present on all segments.
Fourth and fifth pereiopods.Long and slender, exceeding scaphocerite.Propodus of fourth pereiopods with 4-7 (5) pairs of spines distributed along its length, 2× longer than dactylus.Propodus slightly shorter than merus.Ischium shorter than merus.Propodus with fine setae at distal articulation.Scattered short setae present on all segments.Propodus of fifth pereiopods with 4-10 pairs of spines (holotype damaged) distributed along its length and fine setae at distal articulation.Propodus 2.5× longer than carpus.Propodus as long as merus.Scattered short setae present on all segments.
Thoracic sternum.Fourth and fifth thoracic sternites with moderately transverse plate.Sixth to eighth thoracic sternites usually smooth.
Pleon.Smooth.All pleonal sternites with transverse ridge.First and second pleonal sternites with or without median process.Third and fourth pleonal sternites smooth.Fifth sternite with triangular ridge.Preanal carina present, obtuse ridge developed without spine or setae.Ventral margin of pleural tergum with small setae.
Telson (Fig. 6H).Tapered posteriorly, protruding point on middle margin with lateral spines and few fine setae.Inner spines longer than outer spines.Dorsal surface with two pair of small spines, similar in size.
Uropods (Fig. 6H).Uropodal diaeresis with inner movable spine, as long as or slightly longer than outer angle.Exopods longer than endopods.
Etymology.The specific epithet rostrolevatus is from the Latin compound words rostro, for rostrum, and levatus, referring to lifted.
Distribution.This species is distributed in freshwater basins of Khorat Plateau, Northeast Thailand.
Remarks.Macrobrachium rostrolevatus sp.nov.differs from M. lanchesteri s. str.based on the presence of single tooth on movable and fixed fingers of second pereiopods (vs 1 or 2 teeth on movable and fixed fingers in M. lanchesteri), movable spine at uropodal diaeresis slightly longer than the outer angle (vs shorter than outer angle in M. lanchesteri), and the presence of 3-6 pairs of spines on propodus of third pereiopods (vs 4-8 pairs of spines in M. lanchesteri).This new species also differs from M. villosimanus sensu Tiwari (1949) and M. rosenbergii sensu De Man (1879) by having 6-11/4-9 rostral teeth (vs 12-14/7-10 rostral teeth in M. villosimanus; 9-13/10-15 rostral teeth in M. rosenbergii).The second pereiopods are smooth and covered with fine setae (vs spinules in entire cheliped, movable finger densely pubescent and fixed finger sparsely pubescent in M. villosimanus; coarse velvet hairs on movable

Discussion
Morphological and genetic analyses revealed three distinct lineages (prior assumption as geographical variation of M. lanchesteri), which are recognised herein as M. lanchesteri s. str., M. panhai sp.nov., and M. rostrolevatus sp.nov.Previously, the taxonomic identity of M. lanchesteri s. l. was investigated based on the morphological examination of and reinvestigation of type specimens (Lanchester 1902;Chong and Khoo 1988).In this study, the clarification of species boundaries and phylogenetic positions were supplemented by molecular analyses.The phylogenetic position of M. lanchesteri is closely related to M. rosenbergii, although some morphological characteristics might appear similar to the M. sintangense species group.Current observation noted that a juvenile of M. sintangense and M. lanchesteri were morphologically overlapping.Ecologically, they commonly co-exist in several habitats such as riverbanks and lentic reservoirs in mainland Southeast Asia.Their life histories were supposedly influenced by a convergent evolutionary mechanism (Wowor et al. 2009), the same example as noted in other species with abbreviated larval development (ALD) such as Macrobrachium species: M. platycheles Ou & Yeo, 1995, M. sundaicum (Heller, 1862), and M. malayanum (Roux, 1935) (Murphy and Austin 2005).The independent lineages of ALD species were hypothesised as evidence of multiple invasions of marine ancestors (Liu et al. 2007;Murphy and Austin 2005;Wowor et al. 2009).To elucidate the effect of environmental conditions and feeding preferences altering morphological characteristics among coexisting species, comprehensive materials along an environmental gradient could be investigated.Additionally, M. rosenbergii showed distinctiveness in both morphological characters and a reproductive strategy different from M. lanchesteri.The life cycle of M. lanchesteri is completed typically in freshwater as opposed to M. rosenbergii, which had larval development and egg hatching occurring in brackish water.The close phylogenetic relationship between M. lanchesteri and M. rosenbergii seem to potentially derive from a common ancestor through evolutionary divergence processes.
The evidence of genetic divergence and composition differences in Thai invertebrate population are often documented between the lower and upper Isthmus of Kra regions.This evidence was sparsely seen in M. lanchesteri s. str.The same patterns of genetic divergence correlated to subregional populations were also detected in the widespread M. spinipes (Schenkel, 1902).This species shows a wide distribution range in the Indo-Australasian region due to a historical event during the last glacial maximum (De Bruyn and Mather 2007;Ng and Wowor 2011).Currently, the geographical distribution of M. lanchesteri in Southeast Asia seems to possibly include the introduction by human activities, particularly from local fishery-related activities such as in Sabah and Brunei Darussalam (Ng 1994;Wowor and Choy 2001).Thai M. lanchesteri s. str.failed to show a strong subregional pattern despite widespread distribution records, and a similar pattern was also observed in some freshwater gastropods collected from different parts of Thailand (Saijuntha et al. 2021).This might be the consequence of the commercial trade of aquatic plants in Thailand that accidentally introduced freshwater gastropods throughout the area.Contrastingly, M. rostrolevatus sp.nov.has a narrow distribution range and a dense population specifically found in the sub-basins of the Songkhram, Chi, and Mun rivers on the Khorat Plateau.
However, a comprehensive survey of the adjacent sub-basins along the Lower Mekong River Basin should be implemented to affirm its geographic range.
Macrobrachium prawns exhibit a vast variation of morphological characters, with several species demonstrating sexual dimorphism and morphological plasticity (Holthuis 1950;Dimmock et al. 2004;Short 2004).These phenomena increased the uncertainty of species boundaries and the complication of taxonomic discrimination criteria for various Macrobrachium species groups.Recent studies have employed tools, including molecular identification using mitochondrial gene datasets, to clarify and resolve taxonomically ambiguous situations (Liu et al. 2007;Carvalho et al. 2013;Castelin et al. 2017;Rossi et al. 2020;Saengphan et al. 2021).In this study, the mitochondrial genes 16S and COI showed potential to be useful for taxonomic clarification between closely related taxa and revealed the existence of cryptic species, as in the cases of M. panhai sp.nov.and M. lanchesteri s. str.Although M. panhai sp.nov. shares morphological characteristics with M. lanchesteri, genetic differentiation falls within the delimitation gap suggested by Siriwut et al. (2021).For this reason, the delimitation threshold based on inter-and intraspecific variations of Macrobrachium species would be considered an additional tool for cryptic fauna exploration and delineation of morphologically ambiguous groups of Macrobrachium prawns.
Macrobrachium rostrolevatus sp.nov.has different forms of rostrum that appear to be associated with habitat preference.The long and upcurved rostrum is prevalent in lentic habitats i.e., ponds and lakes, whereas the shorter and straight rostrum is dominant in lotic habitats like river tributaries.This rostral shape variability may indicate phenotypic plasticity, similar to observations in M. australe (Guérin-Méneville, 1838) and members of the genus Caridina H. Milne Edwards, 1837, where rostral shape is influenced by water current speed.In an area with fast-flowing current, the long rostrum can be more fragile and impede movement whereas the shorter, more robust, and straight rostrum might better resist the strong water current (Zimmermann et al. 2011;Mazancourt et al. 2017).Moreover, the variation in morphological traits influenced by environment was also found in M. australiensis Holthuis, 1950, an endemic Australian freshwater prawn and M. nipponense (De Haan, 1849), a widespread species in Taiwan (Dimmock et al. 2004;Chen et al. 2015).This study provided additional evidence that the diagnostic characters of Macrobrachium can be influenced by the environment.Therefore, morphological identification alone should be implemented carefully, especially for species with high morphological variability (Liu et al. 2007;Siriwut et al. 2020).The integration of other molecular markers such as nuclear markers and morphometric analysis could be used to further enhance the accuracy of taxonomic identification and phylogenetic relationships of Macrobrachium in the future.

Figure 1 .
Figure 1.Distribution map of three Macrobrachium species in Thailand.A colour symbol indicates the locality of specimen used in phylogenetic analyses.A transparent symbol indicates the locality of specimen examined based on morphology.Equivalent symbols, whether coloured or not, indicate the same species.

Figure 2 .
Figure 2. Phylogenetic tree based on a concatenation of COI and 16S genes.Nodes of a phylogenetic tree marked with a black circle indicate statistical support from both ML and BI (≥ 70 bootstrap values and ≥ 0.95 posterior probability scores).A white circle indicates statistical support for either ML or BI.An asterisk indicates the topotype in M. lanchesteri and holotype in the new species.

Figure 5 .
Figure 5. Morphological characteristics of Macrobrachium panhai sp.nov.(A, F ovigerous female paratype MUMNH MP00303 B-E, G-H ovigerous female holotype CUMZ MP00302) A lateral view B carapace C epistome D second pereiopod E teeth between fingers F third pereiopod G uropod and H movable spine at uropodal diaeresis.Scale bars: 1 mm.

Figure 6 .
Figure 6.Morphological characteristics of Macrobrachium rostrolevatus sp.nov.(A, B, D-I ovigerous female holotype CUMZ MP00323 C ovigerous female specimen MUMNH MP00338.1)A lateral view B carapace C rostral variation D epistome E second pereiopod F teeth between fingers G third pereiopod H uropod and I movable spine at uropodal diaeresis.Scale bars: 1 mm.

Table 1 .
Locality and GenBank accession numbers of specimens used in phylogenetic analyses.