A new species of Eisothistos (Isopoda, Cymothoida) and first molecular data on six species of Anthuroidea from the Peninsular Malaysia

A new species of expanathurid (Eisothistos tiomanensis sp. n.) is described and illustrated. It was collected from Pulau Tioman, Malaysia and can be distinguished by the unique bipartite shape of its uropodal exopod. Accalathura borradailei, Apanthura pariensis, A. stocki, Expanathura corallis and Mesanthura quadrata in the Malaysian waters are recorded for the first time. Additionally, six sequences of cytochrome c oxidase subunit I (COI) genes are presented. These are the first molecular evidence of anthuroids from the waters of Malaysia.


Introduction
The Anthuroidea Leach, 1814 are a distinctive group of mainly marine isopods (Poore 2009).More than a decade ago, Poore (2001) and Brandt and Poore (2003) revised the taxonomic and evolutionary relationships of what had formerly been treated as a suborder, Anthuridea Monod, 1922.In essence, the former work was a cladistics analysis of family and generic relationships which resulted in the recognition of six families while the latter work replaced Anthuridea with the superfamily Anthuroidea within the Cymothoida Wägele, 1989.DNA barcoding was first suggested just over two decades ago (Pecnikar and Busan 2014) and it has been utilized for swift reliable biological identifications.It is a taxonomic method that uses a short genetic marker in an organism's DNA as a unique molecular identifier to a particular species (Hebert et al. 2003a).Hebert et al. (2003b) suggested that the integration of DNA barcoding into traditional taxonomic methods are more efficient in uncovering hidden biodiversity rather than relying on traditional methods alone.On the other hand, due to the increased attention on species barcoding, the issue with questionable and poor quality sequences are becoming more apparent which could possibly harm future systematics studies (Buhay 2009).
Anthuroids have been primarily studied using morphological methods, in particular, dissection of specimens and cladistic analysis.It is only in the recent years that molecular documentations have slowly emerged such as the work of Dreyer and Wägele (2001), Haye et al. (2004), Song andMin (2015a, 2015b) and Wetzer (2001Wetzer ( , 2002)).Although the Malaysian anthuroids have attracted appreciable taxonomic attention lately (Chew et al. 2014;Chew et al. 2016), no molecular data were provided in their studies.The present study is the first to report molecular properties of anthuroids from this region.In order to ensure that the sequences are reliable genes, molecular gene cloning method was implemented and the sequences were assigned to a Phred quality score of 30.

Source of specimens and DNA preservation
The specimens in this study were obtained from coral reef areas around Peninsular Malaysia.Coral rubble was collected into a 56 litres bucket with sea water via SCUBA diving and were moderately broken up.About 10 drops of 37 % formaldehyde were added and left to stand for about 30 minutes.Next, the samples were rinsed and washed with seawater passing through a 500 µm sieve.In the field, samples were fixed with about 10 % formalin in seawater.Molecular specimens were obtained in the same manner except that they were laced with about 1 litre of absolute ethanol and left to stand for about 15 minutes.They were placed directly into precooled absolute ethanol (0 ºC) and kept in an icebox which was carried throughout the field sampling to maximize tissue preservation.

Identification and morphological study
At the laboratory, the specimens were sorted and conserved separately in 4 % formalin in water in vials.Specimens were then identified and new species was selected for dissection.Whole bodies and dissected appendages were mounted in glycerol and illustrated under a Leica DMLB light microscope equipped with a camera lucida.Materials are deposited in the Muzium Zoologi, Universiti Kebangsaan Malaysia, Malaysia.The following abbreviations are used: A, antenna; AM, appendix masculina; MD, mandible; MP, maxilliped; MX, maxilla; P, pereopod; PL, pleopod; PLT, pleotelson U, uropod; UN, uropod endopod; UX, uropod exopod; UKMMZ, Universiti Kebangsaan Malaysia Muzium Zoologi.

DNA extraction, PCR amplification, DNA fragment cloning and sequencing
Before extraction, residual ethanol was removed from the tissue by evaporation at room temperature.The samples were left opened covered only with a Kimwipes tissue until they were completely desiccated.DNA was extracted from whole organism using DNeasy Blood and Tissue Kit (Qiagen, USA).
The COI sequences were amplified using the Folmer et al. (1994) universal primers (LCO1490 and HCO2198).The PCR were performed based on the program: initial denaturation period at 95 ºC for 2 minutes and 35 cycles of denaturation at 95 ºC for 30 seconds, annealing at 48 ºC for 45 seconds and extension at 72 ºC for 90 seconds.A final extension was set at 72 ºC for 5 minutes.PCR products were purified using the Qiaquick PCR purification Kit (Qiagen, USA).
Purified PCR products were cloned using E. coli Top 10 (Invitrogen) and Thermo Scientific CloneJET PCR Cloning Kit (Thermo Fisher Scientific, USA).Colonies containing the vector along with the cloned PCR fragments were picked with a sterile pipette tip and put in to 20 µl of water.This was mixed and 1 µl was taken as the DNA template for PCR colony screening.Successful clones were purified using the innuPREP Plasmid Rapid Kit (Analytik Jena AG, Germany) and sequences were generated on an automated DNA Sequencer ABI 3100 (Applied Biosystems Inc., USA) using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction v3.0 kit (Applied Biosystem Inc., USA).

Bioinformatics screening
Raw COI sequences were assigned to Phred quality score of 30 in order to assess the quality (Ewing and Green 1998).Then, CrossMatch software is used to obtain clean sequences by eliminating the vector sequence (Gotoh 1982).All clones were assembled using the CAP contig assembly program in Bioedit software (Hall 1998).

Results and discussions
Family Anthuridae Leach, 1814 Genus Apanthura Stebbing, 1990 Apanthura forceps Negoescu & Brandt, 2001 Apanthura pariensis Negoescu, 1997 Figure 1 Apanthura pariensis Negoescu, 1997: 186-194  Remarks.Negoescu (1997) described in detail the female and manca materials.Fortunately, the male specimen were collected for the first time in the present study.The materials were found particularly from the west coast of Peninsula Malaysia.This is the first record of such species from this country.
Mouthparts reduced.Pereopod 1 basis 3.3 times as long as greatest width, anterior margin with 1 medial seta, posterior margin with 1 subdistal seta and fine setae; ischium 3.2 times as long  as greatest width, posterior margin subdistally with 1 seta and fine setae; merus anterior margin convex distally, bearing 2 setae, posterior margin with fine setae; carpus anterior margin half the length of posterior margin, posterior margin with 1 seta, with 4 submarginal spinules; propodus 4.4 times as long as greatest width, palm weakly concave with 21 submarginal spinules, anterior margin with 1 distal seta, palmar margin with 1 posterodistal robust seta; unguis half as long as propodus.
Pereopod 2 basis 3.7 times as long as greatest width, posterior margin with 1 subdistal seta and fine setae; ischium 3.1 times as long as greatest width, anterior margin with 2 setae, posterior margin with 2 setae and fine setae; merus anterior margin convex, bearing 1 seta, posterior margin with fine setae; carpus anterior margin half the length of posterior margin, posterior margin with 1 seta and fine setae; propodus 3.9 times as long as greatest width, anterior margin with 2 setae, palmar margin with 1 medial robust seta and 1 posterodistal robust seta, fine setae and subdistal palmar comb; unguis half the length of propodus.
Pereopod 3 basis 3.3 times as long as greatest width, anterior margin with 2 setae and fine setae, posterior mar-gin with 1 seta and fine setae; ischium 3.2 times as long as greatest width, anterior margin with 2 setae and fine setae, posterior margin with 1 seta and fine setae; merus anterior distal margin convex bearing 2 setae, posterior margin with 1 seta, carpus anterior margin one third length of posterior margin, posterior margin with 1 seta; propodus 3.6 times as long as greatest width, anterior margin with 2 setae, palmar margin with fine setae, 2 setae, subdistal palmar comb, 1 medial robust seta and 1 posterodistal robust seta; unguis half as long as propodus.
Pereopod 4 basis 3.9 times as long as greatest width, anterior margin with 2 setae and fine setae, posterior margin with 1 seta and fine setae; ischium 3 times as long as greatest width, anterior margin with 1 seta, posterior margin with 2 setae; merus anterior margin convex, bearing 1 seta, posterior margin with 1 seta, carpus linear twice as long as wide anterior margin with 2 setae, posterior margin with setae comb, 1 seta and 1 robust seta; propodus 4.5 times as long as greatest width, anterior margin with 3 setae, palmar margin bordered with setae comb, 1 medial robust seta and 1 posterodistal robust seta; unguis 0.8 times as long as propodus.
Pereopod 5 basis 4.1 times as long as greatest width, anterior margin with 1 seta, posterior margin with 1 seta and fine setae; ischium 4.1 times as long as greatest width, anterior margin with 1 seta, posterior margin with 3 setae and fine setae; merus anterior margin subdistally convex, bearing 2 setae, posterior margin with fine setae; carpus linear 2.4 times as long as greatest width, anterior margin with 1 seta, posterior margin with setae comb, 1 distal seta and 1 distal robust seta; propodus 4.6 times as long as greatest width, anterior margin with 1 distal seta, palmar margin bordered with setae comb, 1 medial robust seta and 1 posterodistal robust seta; unguis 0.8 times the length of propodus.
Pereopod 6 basis 4 times as long as greatest width, anterior margin with 3 setae and fine setae, posterior margin with 1 subdistal seta; ischium 3.8 times as long as greatest width, anterior margin with fine setae, posterior margin with 3 setae and fine setae; merus anterior margin convex, bearing 1 seta, posterior margin with 1 seta; carpus linear 2.6 times as long as greatest width, anterior margin with 1 seta, posterior margin bordered with setae comb, distally with 1 seta and 1 robust seta; propodus 5.3 times as long as greatest width, palmar margin bordered with setae comb, proximally with 1 robust seta, medially with 1 robust seta and posterodistally with 1 robust seta; unguis 0.7 times the length of propodus.
Pereopod 7 basis 4.1 times as long as greatest width, anterior margin with 1 seta, posterior margin with 1 seta; ischium 3.2 times as long as greatest width, anterior margin with 1 seta and fine setae, posterior margin with 3 setae and fine setae; merus slender 3.6 times as long as greatest width with anterior margin slightly convex, bear-ing 1 seta, posterior margin with 1 seta; carpus linear 3.2 times as long as greatest width, anterior margin with 1 seta, posterior margin with setae comb and 1 robust seta; propodus 7.5 times as long as greatest width, anterior margin with 3 setae, palmar margin bordered with setae comb, 1 medial robust seta and 1 posterodistal robust seta; unguis half as long as propodus.
Pleopod 2 sympod subrectangular; exopod 3 times as long as wide, apex truncated bearing 7 plumose setae; endopod slender 4.8 times as long as greatest width, apex truncated bearing 5 plumose setae; appendix masculina almost as long as endopod, ending in a somewhat hooklike with a rounded end.
Pleopod 5 shorter than others with sympod subquadrate; exopod much smaller than endopod half as long as endopod bearing 3 plumose setae; endopod broadend 1.8 times as long as greatest width bearing 2 plumose setae, apical margin medially with two small protuberances.
Uropod sympod with serrated inner margin, inner distal margin raised with an acute apex, outer distal margin raised with 2 acute serrations apically; endopod elongate with serrated margin bearing 13 setae, apically with 1 short robust plumose sensory spine; exopod bipartite with 5 setae proximally, medially with a central elongated spike having 3 subdistal setae and apical margin concave bearing 1 short plumose sensory spike, outer margin with a short slender spike bearing 1 seta apically, inner margin obsolete.
Pleotelson 2.2 times as long as greatest width, narrowest anteriorly, widest near posterior end, dorsal surface with 4 pairs of setae and a middorsal row of 7 obscure denticles, subdistal margin with 2 obscure teeth on each side, posterior margin strongly dentate with 10 teeth and 5 pairs of setae.
Etymology.This species is named after the type locality, Pulau Tioman, Malaysia.
Molecular data.n/a.

Remarks.
Only one male specimen is available.The male Eisothistos in general, differs from the female by its grossly enlarged eyes, antenna 1 with short basal article bearing numerous aesthetascs, reduced mouthparts, pleonites 1-3 elongate and pleopods 1-3 with elongated peduncles and rami (Knight-Jones and Knight-Jones 2002;Poore 2001;Poore and Lew Ton 2002;Wägele 1979).Though they appear distinctive, their tail-fan shape are more conserved within a species.Hence, it is possible to determine the identity of Eisothistos species based on a male specimen.Moreover in this study, the good condition of preserved material and the unique characteristic of its tail-fan, justify a description.
The uropodal exopod form in the Eisothistos can be divided into two types.While most possess the tripartite uropodal exopod shape, several others are described with the uncommon bipartite uropodal exopod shape.Eisothistos tiomanensis sp.n. belongs to the latter group with Eisothistos anomala, E. corinellae, E. macquariensis and E. minutus (Kensley 1980;Poore and Lew Ton 2002;Sivertsen and Holthuis 1980).It differs in having a unique two slender spine-like structure in the uropodal exopod rather than one central slender spike-like structure along with a proximal lobe as in E. anomala, E. corinellae and E. macquariensis.Eisothistos minutus on the other hand, possesses a form similar to the present species.Sivertsen and Holthuis (1980) described it as "a narrow elongate process on the protopod, it ends in two teeth; the protopod seems to have a second similar process laterally, the nature of this process is not clear".Nevertheless, there is no strong robust seta on the apex of the central spike and a seta on the lateral spike.Additionally, there are no teeth present on the dorsal surface of the pleotelson in E. minutus while there are 7 obscure denticles present in the new Malaysian species.

Distribution.
Pulau Tinggi, Malaysia.Molecular data.A 709 base pairs of COI sequence (Gen-Bank: MF680512) was acquired from one individual of P. tinggiensis.No insertion or deletion in the sequence alignment.