Psammoecus Latreille 1829

© 2018 Academia Sinica, Taiwan Open Access Validation of Male Pontella spinipes Giesbrecht, 1889 (Copepoda: Calanoida: Pontellidae) Based on Morphological and Mitochondrial COI Gene Sequence Analysis Sanu Vengasseril Francis1,*, Shuhei Nishida2, and Sivasankaran Bijoy Nandan1 1Dept. of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and technology, Kerala, India. E-mail: [email protected] (Nandan) 2Atmosphere and Ocean Research Institute, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8564, Japan. E-mail: [email protected] (Received 13 November 2017; Accepted 16 March 2018; Published 17 April 2018; Communicated by Ryuji Machida) Citation: Francis SV, Nishida S, Nandan SB. 2018. Validation of male Pontella spinipes Giesbrecht, 1889 (Copepoda: Calanoida: Pontellidae) based on morphological and mitochondrial COI gene sequence analysis. Zool Stud 57:16. doi:10.6620/ ZS.2018.57-16. Sanu Vengasseril Francis, Shuhei Nishida, and Sivasankaran Bijoy Nandan (2018) The neustonic copepods of the family Pontellidae - Pontella spinipes Giesbrecht, 1889 and P. diagonalis Wilson, 1950, both first described on the basis of female specimens exhibit very similar morphology and overlapping geographic ranges in the Indian Ocean. While several taxonomists have described males of each species, there has been no definitive evidence for female-male matching (link female and male of the same species) in the two species. In the present study, an analysis of mitochondrial cytochrome c oxidase subunit I (mtCOI) sequences in the specimens collected from the Arabian Sea revealed that female P. spinipes Giesbrecht, 1889, and male P. diagonalis sensu Silas and Pillai (1973) are genetically identical, providing evidence that the latter is actually P. spinipes. These findings emphasize that it is necessary to re-examine the female-male correspondence of other related species, formerly based on morphology alone, using molecular-genetic analysis as applied in the present study. Key words: Pontella spinipes, Pontella diagonalis, Mitochondrial cytochrome c oxidase subunit I, Female-male correspondence, Indian Ocean. *Correspondence: E-mail: [email protected] BACKGROUND The calanoid copepod Pontella spinipes Giesbrecht, 1889, was described from the Arabian Sea using female specimens. Wolfenden (1905) described a male from Maldive and Laccadive Islands as P. spinipes but Silas and Pillai (1973) assumed it to be P. diagonalis. Later, the specimens referred to as male P. spinipes were described by Sewell (1912), Silas and Pillai (1973), Pillai (1975), and Mulyadi (2000). Meanwhile, P. diagonalis Wilson, 1950 was also described on the basis of a female specimen from off Jolo Island, the Philippines, and the males referred to as P. diagonalis were described from Indian Ocean by Silas and Pillai (1973), Pillai (1975) and from Indonesian waters by Mulyadi (2000). Researchers believe that there is no distinction between male and females in either species, but there is no morphological evidence to support this claim. Moreover, their geographic ranges, which overlap in the Indian Ocean, and morphological similarity (Wolfenden 1905; Silas and Pillai 1973) arouse our suspicion over the proposed femaleZoological Studies 57: 16 (2018) doi:10.6620/ZS.2018.57-16 1 © 2018 Academia Sinica, Taiwan male matching. The female-male matching means linking female and male of the same species due to the previous descriptions based only on either the female or male specimens or also the morphological resemblance of co-occurring congeners (see also Francis and Nishida 2018). During a taxonomic study on copepods in the coastal waters of the Indian Ocean, we collected female P. spinipes and males assumed to be P. diagonalis by the above authors [hereafter referred to as “male P. diagonalis sensu Silas and Pillai (1973)”], often co-occurring in the same net samples, but did not find female P. diagonalis or males so far assumed to be P. spinipes. Under this circumstance, this study aimed at examining the female-male correspondence of P. spinipes using integrative morphological and molecular genetic analyses. MATERIALS AND METHODS The Pontella copepods were collected during FORV Sagar Sampada cruises in April 2015 from the Arabian Sea (Cruise No. 338, Stations 9, 45 and 48) and in November 2016 from the Bay of Bengal (Cruise No. 353, Stations 2 and 12) (Fig. 1, Table 1) using a plankton net (mesh size: 200 μm; mouth area: 0.28 m2). The net was towed horizontally just below the water surface at a speed of 1 kn for 10 min. For morphological and molecular analysis, the samples were fixed in 4% buffered formalin and 95% ethyl alcohol, respectively. The preservative was changed after 24 h in the alcohol preserved samples. The Pontella specimens for morphological examination were sorted from the original samples; oral parts and swimming legs were dissected in 50:50 solution of glycerine and distilled water. Line drawings were made using a drawing tube attached to a Lynx - bright-field compound microscope (LM 521704). The specimens were identified to species level based on Silas and Pillai (1973). The alcohol-preserved specimens were hydrated in 0.5-mL sterile distilled water for 10-12 hours at room temperature prior to DNA extraction (Sanu et al. 2016). Genomic DNA was extracted from adult individual copepods using the DNeasy Blood & Tissue Kit (Qiagen) following the spin column protocol. The polymerase chain reaction (PCR) mixture consisted of 25 μL Master Mix (Takara Clontech EmeraldAmp® GT PCR Master Mix), 1 μL forward primer, 1 μL reverse primer, 8 μL template DNA, and 15 μL distilled deionized H2O. The amplification primers were LCO-1490 F (5'-GGTC AACAAATCATAAAGATATTGG-3') and HCO-2198 R (5'-TAAACTTC AGGGTGACCAAAAAATCA-3'), used for amplifying mitochondrial cytochrome c oxidase subunit I (mtCOI) gene sequences (Folmer et al. 1994). Amplification was carried out in Agilent technologies thermal cycler (Model no: Sure cycler 8800). The amplification protocol was denaturation at 94°C for 1 min., annealing at 37°C for 2 min. and extension at 72°C for 3 min; 40 cycles were performed. Amplified products exhibiting intense bands after agarose gel (1.2%) electrophoresis was purified and sent to SciGenom Labs (SciGenom Labs Pvt, Ltd. Ernakulam, India) for sequencing. Obtained sequences were assembled using BioEdit 7.0.9 (Hall 1999) and alignment was performed using ClustalX (Thompson et al. 1997). Intraspecific pairwise sequence distance and maximum likelihood tree (ML tree) were estimated using Kimura 2parameter model in MEGA5 (Tamura et al. 2011). Bootstrap analysis was performed using 1000 pseudo replications. Intraspecific aligned sequences were submitted to the National Center for Biotechnology Information (NCBI) database (www.ncbi.nlm.nih.gov). RESULTS SYSTEMATICS Order Calanoida Sars, 1903 Family Pontellidae Dana, 1852 Genus Pontella Dana, 1846 Pontella spinipes Giesbrecht, 1889 Synonymy: Female: Pontella spinipes Giesbrecht, 1889, P. 28; 1892, P. 462, 477, 774, Plate 24 (Fig. 30), Plate 40 (Figs. 2, 23, 24); Wolfenden, 1905, P. 1020-1021; Silas and Pillai, 1973, P. 826-827, Fig. 21a, b, Fig. 22a. Not female Pontella spinipes, Mulyadi, 2011, P. 1523, Fig. 6. Male: Pontella spinipes, Wolfenden, 1905, P. 1020-1021. Pontella diagonalis, Silas and Pillai, 1973, P. 824-826, Fig. 21g, h, l, Fig. 22e (mislabeled as P. securifer); Pillai, 1975, P. 131132, Fig. 1h, i; Mulyadi, 2000, P. 185-186, Fig. 4af, Mulyadi, 2011, P. 1515-1519, Fig.3. Not male Pontella spinipes, Sewell, 1912, P. 373-374, plate 24 (Figs. 1-4); Silas and Pillai, 1973, P. 826-827, Fig. 21c, d, k, Fig. 22b; Pillai, 1975, P. 133-134, Fig. 2a, b; Mulyadi, 2000, P. 193-194, Fig. 13a-d, 2011, P. 1523-1525, Fig. 7. page 2 of 11Zoological Studies 57: 16 (2018) © 2018 Academia Sinica, Taiwan Material examined A total of 150 females and 97 male P. spinipes specimens were collected (Table 1), of which 10 females and 10 males were used for genetic analysis. The other specimens were incorporated into the copepod collection at the Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology (Catalog entry numbers MBM/DBT/13/16 for female MBM/DBT/14/16 for male specimens). Of these, 15 specimens, each of females and males, were examined for the following description. Female: Total length 3.75-4.17 mm (mean: 3.97 mm, n = 15). Body robust (Fig. 2A). Antennule of 23 segments, reaching posterior margin of last pedigerous somite (Fig. 2B). Rostrum bifurcate; upper frontal lens absent; anterior and posterior lower frontal lenses present; diameter of posterior lens 1.3 times that of anterior lens (Figs. 2C, D). Three blue and rounded processes present mid-dorsally on first three pedigerous somites. Fourth and fifth pedigerous somites separate. Posterolateral corners of fifth pedigerous somite produced posteriorly into large pointed lobes, of which left one larger than right and reaching near posterior margin of genital somite. Crescent shaped lobular process present on either side between the pointed lobes of fifth pedigerous somite and insertion of urosome. Urosome two segmented; second segment invisible in dorsal view. Genital somite bulged on its right lateral margin, extending dorso-posteriorly and completely covering second urosomal somite, with small process on right dorsal surface appearing Fig. 1. Sampling locations in the Arabian Sea and Bay of Bengal. Table 1. Summary of sampling data and number of female and male Pontella spinipes collected Cruise Station Latitude (N) Longitude (E) Date (D.M.Y) Number of specimens 338 9 08°15' 28.80" 73°0' 36.00" 14.04.15 67 ♀, 42 ♂ 338 45 12°14' 24.00" 74°19' 12.00" 22.04.15 16 ♀, 5 ♂ 338 48 14°5' 24.00" 73°40' 12.00" 24.04.15 24 ♀, 13 ♂ 353 2 13°5' 14.68" 80°14' 48.12" 15.11.16 7 ♀, 4 ♂ 353 12 15°1' 12.22" 80°12' 43.70" 17.11.16 44 ♀, 33 ♂ page 3 of 11Zoological Studies 57: 16 (2018) © 2018 Academia Sinica, Taiwan Fig. 2. Pontella spinipes, female. (A) Habitus, dorsal view; (B) right antennule; (C) head, right lateral view; (D) rostrum, anterior view, showing anterior and posterior frontal lenses in solid and dotted lines, respectively; (E) urosome, dorsal view; (F) urosome, ventral view; (G) urosome, right lateral view; (H) 5th leg, posterior view. Arrow indicates crescent shaped lobular process present on either side between the pointed lobes of fifth pedigerous somite and insertion of urosome. (A) (B) (C) (D) (F) (E) (G) (H) (H) (A), (B) (C)-(G) page 4 of 11Zoological Studies 57: 16 (2018) © 2018 Academia Sinica, Taiwan as conical projection in right lateral view. Irregular ridges and raised areas present on dorsal surface of genital somite (Fig. 2G). Caudal rami asymmetrical, right ramus larger (Figs. 2E and F). Fifth leg asymmetrical, right leg slightly larger than left; exopod of each leg acuminate and curved with four lateral spinules; endopod about half length of exopod and bifid apically (Fig. 2H). Male: Total length 3.31-3.73 mm (mean: 3.57 mm, n = 15). Fourth and fifth pedigerous somites separate. Posterolateral ends of fifth pedigerous somite produced into symmetrical, acuminate lobes (Fig. 3A). Rostrum well developed with upper frontal lens and anterior and posterior lower frontal lenses (Figs. 3B, C); diameter of anterior lower frontal lens 1.4 times those of upperand posterior lower frontal lenses. Urosome composed of five somites; genital somite asymmetrical with lobe-like projection on left side. Caudal rami nearly symmetrical, each with five long plumose and one small setae. Right antennule (Fig. 3D) geniculate; ancestral segment XIV with stout, elongated spine terminating in bent tapering apex. Fused segments XIX-XX with proximally-oriented scalene-triangular ridge with anterior row of denticles. Fused segments XXIXXIII with anterior process on proximal 1/3, with 3 stout and 2 minute teeth, of which middle one longest (Fig. 3E); distal 2/3 of anterior margin with denticulate plate; segment ends distally in falcate spur. Segments XXIV-XXVIII completely fused. Fifth leg with one seta on each basis. Chela on right leg well developed; outer margin of first exopodal segment with three unequal thumb-like processes basally, of which medial one longest and slightly curved and distal one with basal seta, and semicircular process at midto slightly distal part. Second exopodal segment slender, curved and pointed with two setae near base and seta at distal third. Left leg first exopodal segment with inner medial seta and outer distal spine; second exopodal segment with inner seta near distal third, outer seta near distal third, outer distal seta, two unequal distal spines, of which inner one 1.5 time longer than medial, and two rows of setules along inner margin (Fig. 3F). Remarks: The present female specimens agree with Pontella spinipes Giesbrecht, 1889 as redescribed by Giesbrecht (1982), Wolfenden (1905), and Silas and Pillai (1973), and distinguished from the two closely similar species - P. securifer Brady, 1883 and P. diagonalis Wilson, 1950 - in the following characters: (1) the left pointed lobe of the fifth pedigerous somite is much larger than the right one (the lobes are subequal in P. diagonalis; left one slightly larger than the right one in P. securifer); (2) genital somite bulged on its right lateral margin, extending dorso-posteriorly and completely covering second urosomal somite with a small process on right dorsal surface (on the lateral side of genital somite a conspicuous digitiform process is present in P. securifer, while a sharply pointed, curved process is present in P. diagonalis; see also Tanaka, 1964; Silas and Pillai, 1973; Jeong et al. 2008 for re-descriptions). There have been slight differences between these authors in the number of lateral spinules on the exopod of leg 5: 4 in Giesbrecht (1982) and the present specimens, 3-4 in Silas and Pillai (1973), and 2 in Wolfenden (1905); this may be assumed as intraspecific variation. The female described as P. spinipes by Mulyadi (2011) differs from the present specimens and those described by Giesbrecht (1982) and Silas and Pillai (1973) in (1) the shape of genital somite (much more swollen laterally on the right side than the latters) and (2) the length of the endopod relative to the exopod of the fifth leg (less than 1/4 compared to about 1/2 in the latters. Note that the former length ratio is based on Fig. 6d of Mulyadi (2011) while his text (p. 1523) describes this ratio as about 1/2). As shown in the list of synonymies, male P. spinipes had been described under the name P. diagonalis (Silas and Pillai 1973; Pillai 1975; Mulyadi 2000), except Wolfenden (1905) whose description of male P. spinipes accords with the present specimens, although morphological details of the specimen are unknown since the author did not present any illustrations [note that the figures of male P. diagonalis as described by Mulyadi (2011: Fig. 3) appear to have been copied from Mulyadi (2000: Fig. 4)]. Accordingly, the males described as P. spinipes by Sewell (1912), Silas and Pillai (1973), Pillai (1975), and Mulyadi (2000) are considered to belong to another species and distinguished from true male P. spinipes (= male P. diagonalis sensu Silas and Pillai, 1973) by (1) differences in the shape and size of the processes on the claw of right fifth leg and (2) the size and number of teeth on the elevated process of the fused segments XXI-XXIII of the right antennule (3 conspicuous teeth in P. spinipes) ; note that Silas and Pillai (1973) mislabeled the right antennule of their “male P. diagonalis” as P. securifer (Silas and Pillai 1973: Fig. 22e), as evidenced from their text sentence stating presence of 3 stout subequal teeth on the 19th segment (Silas and Pillai 1973: 825) which accords with their fig. 22e. page 5 of 11Zoological Studies 57: 16 (2018) © 2018 Academia Sinica, Taiwan Fig. 3. Pontella spinipes, male. (A) habitus, dorsal view; (B) head, right lateral view; (C) rostrum, anterior view, showing anterior and posterior lower frontal lenses in solid and dotted lines, respectively; (D) right antennule, number of ancestral segments indicated with Roman numerals; (E) teeth on fused segments XXI-XXIII of right antennule; (F) 5th leg, posterior view. (A) (B) (C) (D) (F) (E) (F) (A) (B), (C) (D), (E) page 6 of 11Zoological Studies 57: 16 (2018) © 2018 Academia Sinica, Taiwan Molecular analysis The mtCOI sequences were successfully generated using the primer pair, reaction mix, and the thermal regime described above. The developed sequences of female P. spinipes and male P. diagonalis sensu Silas and Pillai (1973) were submitted to the NCBI database and assigned the following accession numbers: KT186887 to KT186891and KT267166 to KT267170 for female P. spinipes, KT282363 to KT282372 for male P. diagonalis sensu Silas and Pillai (1973). The base pair length for the developed sequences was 639 bp for the male P. diagonalis sensu Silas and Pillai (1973), and 660 bp for the female P. spinipes. In order to confirm the phylogenetic relationship of these specimens, an ML analysis was performed and pairwise sequence distances were generated and analyzed using the developed sequences as well as the mtCOI sequences of their congeneric species acquired from NCBI database (Table 2). Acartia bispinosa Carl, 1907 was selected as the out-group. The ML tree clearly exhibited the differential assemblage of congeneric species of the genus Pontella (Fig. 4). The female P. spinipes and male P. diagonalis sensu Silas and Pillai (1973) got arrayed within a single clade with the 100% bootstrap value which is distinct from the sequence of P. rostraticauda Ohtsuka, Fleminger and Onbe, 1987 (AB206446). In addition, P. fera Dana, 1849 (KT186882, KT186883) sequences got assembled next to the latter. The clade containing P. sinica Chen and Zhang, 1965 (KT336558, KT336559) and P. chierchiae Giesbrecht, 1889 (JQ714071) is sister to P. fera. As expected, the out-group A. bispinosa (KP068672) exhibited a diverged array. In order to justify the results of the phylogenetic tree, genetic distance persisting within the selected individuals was analyzed. The level of intraand interspecific divergence persisting within the genus Pontella was evident from distance matrix data (Table 3). Specifically, female P. spinipes and male P. diagonalis sensu Silas and Pillai (1973) exhibited 0 - 0.2% intraspecific sequence divergence while all the other selected species showed considerable genetic divergence, justifying the findings of ML tree. DISCUSSION The present genetic analysis demonstrates that the male Pontella specimens described by Silas and Pillai (1973), Pillai (1975), and Mulyadi (2000) under the name Pontella diagonalis actually belong to Pontella spinipes Giesbrecht 1889; the latter was described in detail by Giesbrecht (1982) based on females. A possibility of sequence mismatch between conspecific female and male due to introgression may be ruled out since the sequences’ specimens were collected from multiple distinct areas of the Arabian Sea. Another alternative explanation is that pseudogenes were amplified; this is suspected when two or more sequences are amplified from a single specimen. However, since only a single sequence was amplified from all the specimens examined, there is essentially no possibility of pseudogene amplification. Another possibility, that male P. spinipes and female P. diagonalis have exactly the same sequence, is also highly unlikely considering the generally accepted differences between congeneric copepod species, including those in Pontella, is much greater than ca.10% in COI. Accordingly, the males described as P. spinipes by Sewell (1912), Silas and Pillai (1973), Pillai (1975), and Mulyadi (2000) are considered to belong to another, unknown species. Proving this necessitates the molecular-genetic analysis that the present study applied, which should include Table 2. Details of sequences incorporated and species abbreviations used in the molecular analysis, as applied in table 3 Species Abbreviation Accession numbers Remarks Pontella sinica Chen and Zhang, 1965 PSI KT336559 Obtained P. chierchiae Giesbrecht, 1889 PC JQ714071 Obtained P. fera Dana, 1849 PF KT186882, KT186883 Obtained P. diagonalis sensu Silas and Pillai, 1973 PD ♂ KT282363 to KT282372 Developed P. spinipes Giesbrecht, 1889 PS♀KT186887 to KT186891 and KT267166 to KT267170 Developed P. rostraticauda Ohtsuka, Fleminger and Onbe, 1987 PR AB206446 Obtained Acartia bispinosa Carl, 1907 AB KP068672 Obtained page 7 of 11Zoological Studies 57: 16 (2018) © 2018 Academia Sinica, Taiwan Table 3. Distance matrix showing inter and intraspecific percent divergence of Pontella spinipes and other species in the genus Pontella. See table 2 for specimen and species codes 1234567891011 12 13 1 |KT336559|PSI 2 |KT336558|PS 0.0 3 |JQ714071|PC 20.0 20.0 4 |KT186882|PF 21.9 21.9 23.4 5 |KT186883|PF 22.1 22.1 23.4 0.2 6 |KT282367|PD ♂ 23.2 23.2 23.2 23.5 23.5 7 |KT282368|PD ♂ 23.2 23.2 23.2 23.5 23.5 0.0 8 |KT282366|PD ♂ 23.2 23.2 23.2 23.5 23.5 0.0 0.0 9 |KT282369|PD ♂ 23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 10 |KT282370|PD ♂ 23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 0.0 11 |KT282365|PD ♂ 23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 0.0 0.0 12 |KT282371|PD ♂ 23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 0.0 0.0 0.0 13 |KT282364|PD ♂ 23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 14 |KT282372|PD ♂ 23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 15 |KT282363|PD ♂ 23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 16 |KT267168|PS♀23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 17 |KT267169|PS♀23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 18 |KT267167|PS♀23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 19 |KT267170|PS♀23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 20 |KT267166|PS♀23.2 23.2 23.2 23.5 23.5 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 21 |KT186888|PS♀23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 22 |KT186887|PS♀23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 23 |KT186889|PS♀23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 24 |KT186890|PS♀23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 25 |KT186891|PS♀23.2 23.2 23.2 23.5 23.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 26 |AB206446|PR 21.8 21.8 19.0 23.8 23.8 19.4 19.4 19.4 19.4 19.4 19.4 19.4 19.4 27 |KP068672|AB 29.5 29.5 27.8 30.3 30.5 27.0 27.0 27.0 27.0 27.0 27.0 27.0 27.0 14 15 16 17 18 19 20 21 22 23 24 25 26 27 1 |KT336559|PSI 2 |KT336558|PS 3 |JQ714071|PC 4 |KT186882|PF 5 |KT186883|PF 6 |KT282367|PD ♂ 7 |KT282368|PD ♂ 8 |KT282366|PD ♂ 9 |KT282369|PD ♂ 10 |KT282370|PD ♂ 11 |KT282365|PD ♂ 12 |KT282371|PD ♂ 13 |KT282364|PD ♂ 14 |KT282372|PD ♂ 15 |KT282363|PD ♂ 0.0 16 |KT267168|PS♀0.0 0.0 17 |KT267169|PS♀0.0 0.0 0.0 18 |KT267167|PS♀0.0 0.0 0.0 0.0 19 |KT267170|PS♀0.0 0.0 0.0 0.0 0.0 20 |KT267166|PS♀0.2 0.2 0.2 0.2 0.2 0.2 21 |KT186888|PS♀0.0 0.0 0.0 0.0 0.0 0.0 0.2 22 |KT186887|PS♀0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 23 |KT186889|PS♀0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 24 |KT186890|PS♀0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 25 |KT186891|PS♀0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.0 0.0 26 |AB206446|PR 19.4 19.4 19.4 19.4 19.4 19.4 19.4 19.4 19.4 19.4 19.4 19.4 27 |KP068672|AB 27.0 27.0 27.0 27.0 27.0 27.0 27.0 27.0 27.0 27.0 27.0 27.0 27.5 page 8 of 11Zoological Studies 57: 16 (2018) © 2018 Academia Sinica, Taiwan P. diagonalis females and the females and males that have been identified as closely related to P. securifer. Wolfenden (1905) noted the co-occurrence of a male (= male P. diagonalis sensu Silas and Pillai (1973)) and 3 P. spinipes females in a plankton sample. Silas and Pillai (1973) mentioned the cooccurrence of males (= male P. diagonalis sensu Silas and Pillai (1973)) and females of P. spinipes along with “P. diagonalis”, the gender of which was not specified but may have included males that were really P. spinipes. In the present study we collected Pontella fera Dana, 1849, P. sinica Chen & Zhang, 1965, and P. denticauda A. Scott, 1909 at Stn 9 and P. fera at Stns 2, 12, 45, and 48, along with P. spinipes, many females and males of which were collected together at all stations (Table 1), but P. diagonalis did not occur at all. All these observations are consistent with the present results of female-male correspondence based on genetic information, in that female and male P. spinipes have been collected together in many occasions. It should also be noted that several species of Pontella have often been collected together in same stations and/or plankton-net tows (e.g. Brady 1883; Sewell 1912; Sherman 1964; Silas and Pillai 1973; Mulyadi 2000; this study). This would suggest the presence of mechanisms for co-existence of multiple congeneric species in relatively small areas and the two dimensional habitat in their neustonic life, either by differentiating their habitat water, as defined by various physico-chemical and/or biotic factors (Sherman 1964), and/or food resources (Ohtsuka 1985), inviting further research on their microhabitats and feeding ecology. Fig. 4. Maximum likelihood tree for Pontella spinipes and other Pontella species (taken from GenBank with their accession numbers) based on 1000 bootstrap pseudoreplicas. page 9 of 11Zoological Studies 57: 16 (2018)