Research Article |
Corresponding author: Khwanruan Srinui ( khwanruan@buu.ac.th ) Academic editor: Kai Horst George
© 2019 Khwanruan Srinui, Susumu Ohtsuka, Ephrime B. Metillo, Masahide Nishibori.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Srinui K, Ohtsuka S, Metillo EB, Nishibori M (2019) A new species of Acartia (Copepoda, Calanoida) from the Philippines, based on morphological and molecular analyses. ZooKeys 814: 71-94. https://doi.org/10.3897/zookeys.814.24601
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A new species of Acartia (Odontacartia), A. (O.) edentatasp. n., was collected from Leyte Island in the Philippines. Morphologically, the new species resembles A. (O.) pacifica Steuer, 1915. The female of the new species differs from other species of the A. (O.) erythraea Giesbrecht, 1889 species group in the absence of a pair of sharp spines on the posterior border of the genital double-somite and absence of setules on the lateral margins of urosomites 1–3. Unlike other congeners of the species group, males of the new species lack fine setules along the posterior margin of the prosome. Comparison of the new species with A. (O.) pacifica by pairwise distance data for the 16S (282 bp) gene indicates that these species differ by 20–21%, while the COI gene (636 bp) indicates a difference of 16–17%. The new species seems to be a coastal, occurring in warm waters having a salinity of 33.5.
Acartia , Calanoida , mitochondrial genes, Philippines, phylogeny
The planktonic calanoid copepod genus Acartia Dana, 1846 so far comprises 64 species worldwide (
The centura and erythraea species groups have so far accommodated 7 and 5 species, respectively, with the unassigned species A. (O.) lilljeborgi Giesbrecht, 1889. The centrura species group now accommodates the following seven species (
The common A. (O.) pacifica has a wide distribution on the coasts of the Indo-West Pacific and in East Asian continental waters. The population in the brackish Ariake Sea, western Japan, was identified as a different species, and, after detailed morphological and molecular analyses, was described as A. (O.) ohtsukai Ueda & Bucklin, 2006 and considered to be a continental relict (
During our copepod surveys in southeastern Asia, we found an undescribed species of A. (Odontacartia) from Leyte Island, the Philippines. It is closely related to A. (O.) pacifica, but unique in lacking paired posterodorsal pointed processes on the female genital double-somite. Following
The material examined was collected from three sites: Carigara Bay, Leyte Island, the Philippines (11°30'70"N; 124°69'01"E, depth 15 m) during the daytime on August 23, 2013 (local time 15:55); Ariake Bay, Seto Inland Sea, Pacific Ocean (34°18'40"N; 132°56'40"E, depth 12 m) on August 11, 2011 (local time 13:30); and South Korea (34°40'20"N; 127°48'24"E, depth 24 m) on August 30, 2011 (local time 15:00) (Stations 1–3, respectively, in Fig.
Distribution of Acartia (Odontacartia) pacifica and its sibling species based on data from this and previously published reports. In the present study, samples were obtained from three sites: Station 1, Acartia (Odontacartia) edentata sp. n., Leyte Island, the Philippines (black donut); Station 2, A. (O.) pacifica, Ariake Bay in the Seto Inland Sea, Japan (white donut); and Station 3, A. (O.) pacifica, South Korea Sea, Korea. Data from earlier studies indicate the distribution of A. (O.) pacifica in the Pacific Ocean (
The structure of female and male antennules follows a pattern of the basically uniramous 28 segments. The antennules of both sexes are similar except for the geniculate right antennule in calanoid copepods as in the copepodid I (CI) setation pattern (
In this study, we used adults of the undescribed species Acartia (O.) from the Philippines and A. (O.) pacifica individuals for genetic analysis of the mitochondrial cytochrome oxidase I (COI) and 16S rRNA (16S) genes. DNA for PCR amplification was prepared from individual males or females placed in microcentrifuge tubes with 50 µl chelex 5%, 1 µl Proteinase K (20 mg/ml). Tubes were heated to 65 °C for 1 hour, boiled at 100 °C for 8 min, and centrifuged at 10,000 x g for 8 min. PCR reagents included 5 µl of 10× buffer, 4 µl of 50 mM MgCl2, 5 µl of 2 mM dNTPs, 0.25 µl of 10 µM primer solutions, 0.25 µl Taq DNA polymerase (Product no. PL1202, Vivantis, Malaysia) and 30.25 µl distilled water, following (
DNA sequences were manually edited using Sequence Scanner version 1.0 (Applied Biosystems) and compared with the GenBank: A. (O.) pacifica (accession number DQ071177 for COI and DQ071175 for 16S); A. (O.) ohtsukai (accession no. DQ071177 for COI and DQ071176 for 16S); Acartia (Acanthacartia) tsuensis Itô, 1956 (accession no. KC287427 for COI); and A. (O.) erythraea (accession no. DQ320504 for 16S). Sequences and multiple alignments were constructed with BioEdit version 7.1 (
Type locality: Carigara Bay, Off Leyte Island, the Philippines (11°30'70"N; 124°69'01"E) (Fig.
Holotype: ♀, dissected and mounted on 2 glass slides (BIMS–Zoo–0267); paratype (allotype): 1 ♂, dissected and mounted on 2 glass slides (BIMS–Zoo–0268); additional paratypes: 4 ♀, 3 ♂ partially dissected and mounted on 3 glass slides (BIMS–Zoo–0269).
Female. Total length, 1.19–1.23 mm (mean ± SD = 1.21 ± 0.01 mm, N = 10; holotype, 1.19 mm); prosome length, 0.42–0.46 mm (0.44 ± 0.01 mm; holotype, 0.44 mm); prosome width, 0.24–0.29 mm (0.26 ± 0.01 mm; holotype, 0.25 mm). Male. Total length 1.08–1.15 mm (mean ± SD = 1.10 ± 0.02 mm, N = 10; allotype, 1.10 mm); prosome length, 0.39–0.41 mm (0.40 ± 0.00 mm; allotype, 0.40 mm); prosome width, 0.23–0.26 mm (0.24 ± 0.01 mm; allotype, 0.25 mm).
Female. Body (Fig.
Antennule (Fig.
Antenna (Fig.
Mandible (Fig.
Maxillule (Fig.
Maxilla (Fig.
Maxilliped (Fig.
Legs 1 to 4 (Fig.
Acartia (Odontacartia) edentata sp. n. armature formula for legs 1–4, with spines and setae indicated by Roman and Arabic numerals, respectively, following
Coxa | Basis | Exopod segment | Endopod segment | ||||
---|---|---|---|---|---|---|---|
1 | 2 | 3 | 1 | 2 | |||
Leg 1 | 0-0 | 0-0 | 1-1; I-1; 2,I,4 | 0-1; 1,2,3 | |||
Leg 2 | 0-0 | 0-0 | 0-1; 0-1; 0,I,5 | 0-2; 1,2,4 | |||
Leg 3 | 0-0 | 0-0 | 0-1; 0-1; 0,I,5 | 0-2; 1,2,4 | |||
Leg 4 | 0-0 | 1-0 | 0-1; 0-1; 0,I,5 | 0-3; 1,2,3 |
Leg 5 (Fig.
Male. Body (Fig.
Left antennule (Fig.
Right antennule (Fig.
Leg 5 (Fig.
The absence of paired dorsal processes on the female genital double-somite and the thick rostrum were confirmed with scanning electron microscopy (Fig.
SEM observations of the male urosomite clearly showed fine ornamentation on the posterior border of first urosomite (Fig.
The subgenus Acartia (Odontacartia) is composed of two species groups, the centrura and erythraea species groups (
Among the centrura species, the female of A. (O.) edentata sp. n. is unique in lacking paired posterior dorsolateral processes on the genital double-somite unlike those of the closely related A. (O.) pacifica (Table
Differences in morphological characteristics among Acartia (Odontacartia) edentata sp. n., A. (O.) pacifica from Japan and Korea, and A. (O.) ohtsukai.
Features | A. (O.) edentata sp. n. | A. (O.) pacifica (Japan and Korea) | A. (O.) ohtsukai* |
---|---|---|---|
Female | |||
Setae on segment (5) VII of right antennule | 1 seta | Absent | 1 seta |
Paired posterior dorsolateral processes on the genital double-somite | Absent | Present | Present |
Length of dorsal processes on the second urosomite relative to the posterior border of the anal somite | Reaching posterior border of anal somite | Half of length | Half of length |
Length ratio of lateral to terminal setae of leg 5 | 1.3 | 2 | 1 |
Mandibular processes | 1 blunt and 2 cuspidate | 1 blunt covered with chitosan and 6 cuspidate | 5 cuspidate |
Male | |||
Length of dorsal and lateral spines on second somite | Medium | Long | Short |
Presence of three rows of spinules ventrolaterally on second somite | Present | Present | Absent |
Dorsal processes of third urosomite long enough to reach beyond those of fourth urosomite | Reaching | Not reaching | Not reaching |
Insertion of inner seta on second exopod segment of left leg 5 | Midway | Subterminal | Subterminal |
Shape of medial projection on second exopodal segment of right leg 5 | Irregular triangular | Rounded triangular | Quadrate |
Since “A. (O.) pacifica” s.l. morphologically and genetically consists of several cryptic species (
The taxonomy of the Indo-West Pacific A. (O.) pacifica should be revised, because the presence of several cryptic species has already been suggested by our study and others. The above-mentioned sexual dimorphic features are species-specific, and should be carefully compared among A. (O.) pacifica s.l. to resolve the issue (see Discussion).
The new species of Acartia was named edentata (Latin, meaning toothless) with reference to the absence of tooth-like processes on the posterodorsal border of the genital double-somite in females.
We obtained sequence data from mitochondrial 16S and COI genes for 14 individual specimens at three sites. A 282 bp fragment of the 16S gene was analyzed for five adult female specimens from the Philippines (A. (O.) edentata sp. n.), and a 162 bp 16S fragment was analyzed for A. (O.) pacifica specimens from Ariake Bay, the Seto Inland Sea, Japan and South Korea. A 636 bp fragment of the mitochondrial COI gene was analyzed in the new species and in specimens from Japan and Korea. GenBank sequences for A. (O.) pacifica (accession number DQ071175 for 16S and DQ071177 for COI) and two out group species of subgenus A. (Odontacartia), A. (O.) ohtsukai (accession number DQ071174 for 16S and DQ071176 for COI), A. (O.) erythraea (accession number DQ320504 for 16S) and A. (O.) tsuensis (accession number KC287427 for COI), were also used for comparison. The intraspecific variation in the 16S sequences from the five A. (O.) edentata sp. n. individuals was 0%, whereas A. (O.) edentata sp. n. sequences differ from those of A. (O.) pacifica from Japanese and Korean waters, A. (O.) pacifica based on GenBank, A. (O.) ohtsukai, and A. (O.) erythraea by 20–21%, 20–21%, 28%, and 31%, respectively. The COI sequences from A. (O.) edentata sp. n. individuals differ by only 0.02–0.08%; A. (O.) pacifica (from Japan, and Korea), A. (O.) pacifica (GenBank), A. (O.) ohtsukai, and A. (O.) tsuensis sequences differ from A. (O.) edentata sp. n. COI sequences by 16–18%, 16–17%, 16–17%, 22%, and 24%, respectively (Fig.
Neighbor-joining phylogenetic tree based on the mitochondrial 16S (A) and COI (B) genes of Acartia (Odontacartia) edentata sp. n., and A. (O.) pacifica from Ariake Bay in the Seto Inland Sea and Korean waters. A. (O.) erythraea, A. (O.) ohtsukai and A. (O.) tseuensis sequences from GenBank were used as outgroups. Bootstrap values (percentage) are shown for nodes with support > 50%. Supporting valves of each node obtained from 1,000 bootstrap replications.
Pairwise differences for 16S and COI sequences between individual females of Acartia (Odontacartia) edentata sp. n. from Leyte Island, the Philippines; A. (O.) pacifica from Ariake Bay, Seto Inland Sea (GenBank accession no. DQ071175 for 16S and DQ071177 for COI); A. (O.) pacifica from South Korea and Japan (Seto Inland Sea); A. (O.) ohtsukai from the Rokkaku River Estuary, Ariake Bay (GenBank accession no. DQ071174 for 16S and DQ071176 for COI); A. (O.) erythraea (GenBank accession no. DQ320504 for 16S); and A. (A.) tsuensis (GenBank accession no. KC287427 for COI).
16S | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
1 | Acartia edentata | ||||||||||||||||
2 | Acartia edentata | 0.000 | |||||||||||||||
3 | Acartia edentata | 0.000 | 0.000 | ||||||||||||||
4 | Acartia edentata | 0.000 | 0.000 | 0.000 | |||||||||||||
5 | Acartia edentata | 0.000 | 0.000 | 0.000 | 0.000 | ||||||||||||
6 | Acartia pacifica (Japan1) | 0.206 | 0.206 | 0.206 | 0.206 | 0.206 | |||||||||||
7 | Acartia pacifica (Japan3) | 0.206 | 0.206 | 0.206 | 0.206 | 0.206 | 0.006 | ||||||||||
8 | Acartia pacifica (Japan4) | 0.219 | 0.219 | 0.219 | 0.219 | 0.219 | 0.019 | 0.013 | |||||||||
9 | Acartia pacifica (Japan 5) | 0.212 | 0.212 | 0.212 | 0.212 | 0.212 | 0.013 | 0.006 | 0.019 | ||||||||
10 | Acartia pacifica (Korea1) | 0.200 | 0.200 | 0.200 | 0.200 | 0.200 | 0.025 | 0.019 | 0.031 | 0.025 | |||||||
11 | Acartia pacifica (Korea2) | 0.200 | 0.200 | 0.200 | 0.200 | 0.200 | 0.025 | 0.019 | 0.031 | 0.025 | 0.000 | ||||||
12 | Acartia pacifica (Korea3) | 0.200 | 0.200 | 0.200 | 0.200 | 0.200 | 0.025 | 0.019 | 0.031 | 0.025 | 0.000 | 0.000 | |||||
13 | Acartia pacifica (Korea4) | 0.200 | 0.200 | 0.200 | 0.200 | 0.200 | 0.025 | 0.019 | 0.031 | 0.025 | 0.000 | 0.000 | 0.000 | ||||
14 | Acartia pacifica (Korea5) | 0.212 | 0.212 | 0.212 | 0.212 | 0.212 | 0.013 | 0.006 | 0.019 | 0.013 | 0.013 | 0.013 | 0.013 | 0.013 | |||
15 | Acartia pacifica (DQ 071175) | 0.206 | 0.206 | 0.206 | 0.206 | 0.206 | 0.006 | 0.000 | 0.013 | 0.006 | 0.019 | 0.019 | 0.019 | 0.019 | 0.006 | ||
16 | Acartia ohtsukai (DQ071176) | 0.287 | 0.287 | 0.287 | 0.287 | 0.287 | 0.287 | 0.281 | 0.281 | 0.287 | 0.275 | 0.275 | 0.275 | 0.275 | 0.281 | 0.281 | |
17 | Acartia erythraea (DQ320504) | 0.313 | 0.313 | 0.313 | 0.313 | 0.313 | 0.356 | 0.350 | 0.338 | 0.350 | 0.344 | 0.344 | 0.344 | 0.344 | 0.350 | 0.350 | 0.331 |
COI | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | |
1 | Acartia edentata | ||||||||||||||||
2 | Acartia edentata | 0.008 | |||||||||||||||
3 | Acartia edentata | 0.002 | 0.009 | ||||||||||||||
4 | Acartia edentata | 0.008 | 0.003 | 0.009 | |||||||||||||
5 | Acartia edentata | 0.000 | 0.008 | 0.002 | 0.008 | ||||||||||||
6 | Acartia pacifica (Japan1) | 0.173 | 0.178 | 0.175 | 0.178 | 0.173 | |||||||||||
7 | Acartia pacifica (Japan3) | 0.176 | 0.181 | 0.178 | 0.181 | 0.176 | 0.019 | ||||||||||
8 | Acartia pacifica (Japan4) | 0.164 | 0.165 | 0.165 | 0.165 | 0.164 | 0.017 | 0.020 | |||||||||
9 | Acartia pacifica (Japan5) | 0.175 | 0.180 | 0.176 | 0.180 | 0.175 | 0.017 | 0.011 | 0.019 | ||||||||
10 | Acartia pacifica (Korea1) | 0.162 | 0.167 | 0.164 | 0.167 | 0.162 | 0.024 | 0.024 | 0.022 | 0.022 | |||||||
11 | Acartia pacifica (Korea2) | 0.165 | 0.170 | 0.167 | 0.170 | 0.165 | 0.030 | 0.030 | 0.028 | 0.028 | 0.009 | ||||||
12 | Acartia pacifica (Korea3) | 0.162 | 0.167 | 0.164 | 0.167 | 0.162 | 0.024 | 0.024 | 0.022 | 0.003 | 0.022 | 0.006 | |||||
13 | Acartia pacifica (Korea4) | 0.164 | 0.169 | 0.165 | 0.169 | 0.164 | 0.025 | 0.025 | 0.024 | 0.024 | 0.005 | 0.008 | 0.002 | ||||
14 | Acartia pacifica (Korea5) | 0.161 | 0.165 | 0.162 | 0.165 | 0.161 | 0.025 | 0.025 | 0.024 | 0.024 | 0.011 | 0.020 | 0.014 | 0.016 | |||
15 | Acartia pacifica (DQ 071177) | 0.167 | 0.172 | 0.169 | 0.172 | 0.167 | 0.009 | 0.009 | 0.011 | 0.008 | 0.014 | 0.020 | 0.014 | 0.016 | 0.016 | ||
16 | Acartia ohtsukai (DQ071176) | 0.220 | 0.220 | 0.222 | 0.220 | 0.220 | 0.241 | 0.241 | 0.233 | 0.239 | 0.231 | 0.235 | 0.231 | 0.233 | 0.235 | 0.231 | |
17 | Acartia tsuensis (KC287427). | 0.243 | 0.244 | 0.244 | 0.244 | 0.243 | 0.247 | 0.252 | 0.244 | 0.249 | 0.250 | 0.249 | 0.247 | 0.249 | 0.252 | 0.243 | 0.247 |
Temperature and salinity appear to be important factors determining the distribution and abundance of copepods. In the Indo-West Pacific, A. (O.) pacifica occurs in the tropical and subtropical zones of the Pacific and Indian oceans. In the East China Sea (subtropical zone), A. (O.) pacifica was abundant in August (salinity 15.0) in the Changjiang (Yangtze River) Estuary, China (
In the tropical zone, A. (O.) edentata sp. n. specimens were collected in the Philippines during the rainy season (August 2013), when water temperature and salinity were 30.2 °C and 33.5, respectively. In contrast, A. (A.) tsuensis represents the dominant species in brackish pond water from Panay Island in central Philippines during the dry season (November – April), with salinity ranging from 14.0 to 40.0 (
In Bintulu, Sarawak, Malaysia,
Prior to the current study, it was believed that A. (O.) pacifica was represented by a single species with a wide geographic range occupying the coastal brackish waters throughout the Western Pacific and Indian oceans (
Mitochondrial markers within the 16S and COI genes have proved to be of great utility in investigating the systematics of ecologically and geographically isolated populations of calanoid copepods (
Thirteen species of the subgenus Acartia (Odontacartia), including A. (O.) edentata sp. n., have been described from the Indo-West Pacific (
Female
1 | Genital double-somite lacking posterodorsal sharp processes | 2 |
– | Genital double-somite having paired posterodorsal processes | 3 |
2 | Ventroposterior corners of prosome acutely pointed, reaching beyond half of genital double-somite | A. (O.) edentata sp. n. |
– | Ventroposterior corners of prosome round with pair of acutely pointed processes not reaching beyond half of genital double-somite | A. (O.) bowmani |
3 | Second segment of antennule with strong curved processes posteriorly | 4 |
– | Second segment of antennule without strong curved processes | 5 |
4 | First antennule segment with two large processes terminally | A. (O.) bispinosa Carl, 1907 |
– | First antennule segment lacking processes | A. (O.) spinicauda |
5 | Exopod of leg 5 thickened proximally | 6 |
– | Exopod of leg 5 not thickened proximally | 8 |
6 | Exopod of leg 5 thickened proximally extending midway along exopod | A. (O.) centrura |
– | Exopod of leg 5 with thickened proximal part confined to base of exopod | 7 |
7 | Length ratio of outer basal setae to exopod of leg 5: ca 2 | A. (O.) pacifica |
– | Length ratio of outer basal setae to exopod, leg 5: ca 1 | A. (O.) ohtsukai |
8 | Caudal ramus longer than wide by at most ca 2 times; second free urosomite with small spinules dorsally and posteriorly | 9 |
– | Caudal ramus longer than wide by ca 3 times; second free urosomite lacking small dorsal spinules | A. (O.) mertoni |
9 | Fifth to seventh antennule segments each with posterior hook; genital double-somite with two pairs of small processes dorsally | A. (O.) lilljeborgi |
– | Fifth to seventh antennule segments each lacking hook posteriorly; genital double-somite with pair of small processes dorsally | 10 |
10 | First antennule segment with 2 or more strong processes distally | 11 |
– | First antennule segment with single strong process distally | 12 |
11 | Second antennule segment with single spinule posteriorly | A. (O.) erythraea |
– | Second antennule segment with 4 spinules posteriorly | A. (O.) amboinensis Carl, 1907 |
12 | Caudal ramus with 4–6 rows of minute spinules dorsally | A. (O.) japonica Mori, 1940 |
– | Caudal ramus lacking of dorsal rows of spinules | A. (O.) australis Farran, 1936 |
Male
1 | Urosomite 3 with large spine-like processes dorsally | 2 |
– | Urosomite 3 without spine-like processes dorsally | 9 |
2 | Dorsal processes of urosomite 3 long, reaching half-length of anal somite | 3 |
– | Dorsal processes of urosomite 3 short, reaching posterior-most border of urosomite 4 | 6 |
3 | Urosomite 4 with four spine-like processes between pair of dorsal processes | A. (O.) spinicauda |
– | Urosomite 4 lacking spine-like processes between pair of dorsal processes | 4 |
4 | Genital somite lacking spinular rows along posterodorsal border | A. (O.) pacifica |
– | Genital somite with spinular rows along posterodorsal border | 5 |
5 | Inner projection of first exopodal segment of right leg 5 quadrate | A. (O.) mertoni |
– | Inner projection of first exopodal segment of right leg 5 irregularly triangular | A. (O.) edentata sp. n. |
6 | Urosomites 3 and 4 each with two prominences between pair of dorsal processes | A. (O.) centrura |
– | Urosomites 3 and 4 each lacking prominences between pair of dorsal processes | 7 |
7 | Inner seta of terminal exopodal segment of left leg 5 longer than terminal segment | A. (O.) ohtsukai |
– | Inner seta of terminal exopodal segment of left leg 5 nearly equal to terminal segment | A. (O.) bowmani |
8 | Urosomite 4 without prominences dorsally | A. (O.) australis |
– | Urosomite 4 with prominences dorsally | 9 |
9 | Number of dorsal prominences on urosomite 4 fewer than five | 10 |
– | Number of prominences on urosomite more than seven | 12 |
10 | Terminal exopodal segment of left leg 5 with three elements | A. (O.) erythraea |
– | Terminal exoposal segment of left leg 5 with single element | 11 |
11 | Terminal element of left leg 5 spiniform | A. (O.) amboinensis |
– | Terminal element of left leg 5 as fine seta | A. (O.) lilljeborgi |
12 | Terminal elements of left leg 5 as three small prominence | A. (O.) japonica |
– | Terminal elements of left leg 5 as two spines | A. (O.) bispinosa |
We express our sincere thanks to Dr Mitsuyasu Moriya for his help with field sampling at Leyte Island in the Philippines. We are also grateful to Dr Wansuk Senanan of Burapha University for guidance on molecular techniques. Thanks are also due to Professor Ho Young Soh (Chonnan National University, Korea) who kindly provided us with specimens of Acarita (Odontoacartia) pacifica collected from Korea. This study was partially supported by grants from the Asian CORE-to-CORE and RONPAKU Programs of the Japan Society for the Promotion of Science (JSPS), a KAKEN grant from the JSPS (awarded to SO, 16K07825), and by a grant from Burapha University (to KS, 1/2559).